Autoimmune responses to the β cell autoantigen, insulin, and the INS VNTR-IDDM2 locus

Abstract

SUMMARYType 1 diabetes is associated with autoimmunity to insulin. Genetic susceptibility to type 1 diabetes is polygenic and includes the INS VNTR-IDDM2 locus which may regulate the expression of insulin in pancreas and thymus. In order to determine whether insulin autoimmunity could be attributed to a genetic susceptibility conferred by the INS VNTR-IDDM2 locus, peripheral blood T cell proliferation to human insulin and insulin autoantibodies (IAA) was measured in patients with new onset type 1 diabetes and control subjects. IAA were detected in 21 of 53 patients and in none of 25 control subjects, while T cell responses were low (stimulation index range 0·4-7·2) and similar in both groups. Both antibody and T cell responses were higher in younger subjects and IAA were more prevalent in patients with the HLA-DR4 allele. No relationship was observed between humoral and cellular responses to insulin. No association was found between the INS VNTR-IDDM2-susceptible allele and insulin autoimmunity. Increased T cell responses and IAA were found in patients with either the diabetes-susceptible or the diabetes-protective INS VNTR-IDDM2 locus genotypes, and increased T cell responses were also found in control subjects with either susceptible or protective INS VNTR-IDDM2 locus genotypes. This study confirms that primary T cell proliferative responses to insulin are low and detectable also in control subjects. The detection of T cell proliferation and autoantibodies to insulin in subjects with and without the protective INS VNTR-IDDM2 locus genotypes does not support the hypothesis of an allele-specific capacity for tolerance induction which could determine a susceptibility to develop autoimmunity against the insulin protein and subsequently diabetes.

A disease-associated cellular immune response in type 1 diabetics to an immunodominant epitope of insulin

“…Peptides, reagents, and tissue culture medium. The following peptides were synthesized by a solid-phase method as described elsewhere (24): human insulin B-chain (9-23) amide (B (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) ) [SHLVEALYLVCGERG], sperm whale myoglobin 110-121 (SWM), and biotinylated herpes simplex virus peptide-2 (HSV-2) VP16 430-444 (EEVDMTPADALDDFD). Medium used for all cell cultures and assays was DMEM containing high glucose (Cellgro, Herndon, Virginia, USA) supplemented with 2 mM L-glutamine, 10 mM HEPES (Cellgro), nonessential amino acids (Sigma Chemical Co., St Louis, Missouri, USA), 1 mM sodium pyruvate, 50 µg/ml gentamicin, 126 µg/ml L-arginine, 20 µg/ml L-aspartic acid (Life Technologies Inc., Grand Island, New York, USA), 50 µM 2-Mercaptoethanol (Sigma Chemical Co.), 5 µg/ml penicillin and 100 U/ml streptomycin (Life Technologies Inc.), and 5% autologous human serum or plasma.…”

“…PBMCs from all subjects were isolated by centrifugation through Ficoll-Hypaque density gradients (Vacutainer CPT; Becton Dickinson Labware, Lincoln Park, New Jersey, USA), and lymphocytes were washed and either stimulated with B (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) peptide to generate Tcell lines or stored frozen in aliquots for future evaluation of cytokine production using the ELISPOT assay. Insulin B (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) -specific T-cell lines were generated by culturing 5 × A Serum levels of anti-GAD65 and anti-IA-2 antibody from type 1 diabetic patients (P#) and prediabetic individuals at high risk for diabetes who have not received insulin therapy (H#) were measured by a liquid-phase competitive RIA and anti-insulin levels were measured by a protein A microassay. Positive and negative control sera were included that were used to calculate an index for antibody levels as described by the following equation: (unknown sample valuenegative control value)/(positive control value -negative control value).…”

“…Rather, the disease is predominantly mediated by autoreactive cellular responses to βCAs (2). Unexpectedly, however, the T-cell proliferative responses to proinsulin or to the whole insulin protein do not appear elevated in prediabetic patients or in those with recent-onset type 1 diabetes compared with normal control subjects (12)(13)(14)(15)(16)(17)(18).…”

“…Therefore, we addressed whether a more restricted-epitope response to insulin is also characteristic of human type 1 diabetes by determining whether elevated insulin B (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23) -specific cellular responses exist in prediabetic and recent-onset type 1 diabetic patients.…”

The effect of HLA class II, insulin and CTLA4 gene regions on the development of humoral beta cell autoimmunity

“…An early study observed no association between the INS VNTR genotype and insulin-specific cellular and humoral autoimmunity [19], while class I alleles of the VNTR were found to contribute to the emergence of IAA in a larger case-control comparison [7]. It has also been suggested that the INS locus confers increased disease risk only in subjects with moderate-risk HLA genotypes [20].…”