Mature high-affinity immune responses to (pro)insulin anticipate the autoimmune cascade that leads to type 1 diabetes

Abstract

Children at risk for type 1 diabetes can develop early insulin autoantibodies (IAAs). Many, but not all, of these children subsequently develop multiple islet autoantibodies and diabetes. To determine whether disease progression is reflected by autoantibody maturity, IAA affinity was measured by competitive radiobinding assay in first and subsequent IAA-positive samples from children followed from birth in the BABYDIAB cohort. IAA affinity in first positive samples ranged from less than 10 6 l/mol to more than 10 11 l/mol. High affinity was associated with HLA DRB1*04, young age of IAA appearance, and subsequent progression to multiple islet autoantibodies or type 1 diabetes. IAA affinity in multiple antibody-positive children was on average 100-fold higher than in children who remained single IAA positive or became autoantibody negative. All high-affinity IAAs required conservation of human insulin A chain residues 8-13 and were reactive with proinsulin. In contrast, most lower-affinity IAAs were dependent on COOH-terminal B chain residues and did not bind proinsulin. These data are consistent with the concept that type 1 diabetes is associated with sustained early exposure to (pro)insulin in the context of HLA DR4 and show that high-affinity proinsulin-reactive IAAs identify children with the highest diabetes risk.

GAD autoantibody affinity in schoolchildren from the general population

“…Interestingly, GADA binding to the middle and/or C-terminal part of GAD65 could be of high or low affinity, and this was strongly related to the islet autoantibody status. The occurrence of GADA plus IAA and/or IA-2A was predominantly seen in children with high-affinity autoantibodies, confirming previous reports from us and others [25,27,28]. In contrast, GADA binding only to the N-terminal part of GAD65 involved predominantly single low-affinity GADA level (KU/l) GADA C-terminal level (AU) Fig.…”

In insulin-autoantibody-positive children from the general population, antibody affinity identifies those at high and low risk

“…1b). As in findings in IAA-positive first-degree relatives [7], IAA affinity of ≥10 9 l/mol was a discriminatory threshold: 22 (92%) of the 24 children who developed other islet autoantibodies or diabetes had IAA affinities ≥10 9 l/mol, compared with only four (18%) of 22 of the children who did not progress to multiple islet autoantibodies or diabetes (p<0.0001).…”

“…For early risk assessment, therefore, it would be useful to identify IAA-positive children who will develop other islet autoantibodies. We showed that high IAA affinity and proinsulin reactivity were characteristics of IAA in first-degree relatives who developed further islet autoantibodies and type 1 diabetes [7]. The purpose of this study was to extend these findings to children from the general population.…”

“…IAA affinity and epitope analysis IAA affinity was measured in blinded fashion on coded sera by competitive inhibition in a protein A/G radiobinding assay, as previously described [7]. Antibody binding to 7.85 fmol of human Tyr 14 A [ 125 I] insulin (Aventis, Frankfurt, Germany; specific activity 370 MBq/g; 0.128 nmol/l) was competed with five increasing concentrations of unlabelled human insulin (Insuman Rapid; Aventis; 5.6×10 −6 , 2.8×10 −7 , 2.8×10 −8 , 1.7×10 −9 , 5.5×10 −11 mol/l) in a total volume of 55 μl per tube.…”

“…In order to determine the proinsulin reactivity of IAA, human proinsulin (Eli Lilly, Indianapolis, IN, USA; 1.0×10 −7 mol/l) was used for competition. IC 50 and K d values were calculated using the GraphPad Prism3 program (GraphPad Software, San Diego, CA, USA), and IAA affinity was expressed as reciprocal K d values (l/mol) as previously described [7].…”