Mature high-affinity immune responses to (pro)insulin anticipate the autoimmune cascade that leads to type 1 diabetes (original) (raw)
IAA binding characteristics are consistent with a 1-site binding model and therefore relatively homogeneous within samples. IAAs are measured by radiobinding assay using [125I] insulin labeled at tyrosine at position 14 of the A chain. To determine whether iodine labeling at this position affected the binding of autoantibodies and therefore IAA affinity measurements, [125I] insulin labeled at 1 of 3 different tyrosine residues of the insulin A chain (Tyr14A, Tyr19A) or B chain (Tyr16B) were used to measure affinity in an IAA-positive relative. Although the nonspecific binding was increased when Tyr16B insulin was used as label, binding curves and IC50 and _K_d values were similar with the [125I] insulin labeled at residues Tyr14A, Tyr19A, or Tyr16B (Figure 1A), indicating that Tyr14A iodine labeling is unlikely to interfere with the binding of IAAs.
Competitive insulin binding curves of IAAs. (A) Competition of an IAA-positive serum against [125I] insulin labeled at Tyr14A (circles), Tyr19A (triangles), and Tyr16B (diamonds) with increasing concentrations of unlabeled human insulin. Binding curves were similar with the [125I] insulin labeled at residues Tyr14A, Tyr19A, or Tyr16B, and calculated IAA affinities did not significantly differ among Tyr14A [125I] insulin (2.1 × 1011 l/mol), Tyr19A [125I] insulin (2.3 × 1011 l/mol), and Tyr16B [125I] insulin (5.7 × 1011 l/mol). Insulin labeled at position Tyr16B was associated with considerably higher nonspecific binding than insulin labeled at Tyr14A or Tyr19A. (B) Scatchard analysis performed for the competition curve obtained against Tyr14A radiolabeled insulin. (C) Binding curves of a serum mix containing a serum with high-affinity IAAs (1.7 × 1011 l/mol) and a serum with low-affinity IAAs (2 × 105 l/mol) (squares) and a serum mix containing a serum with high-affinity IAAs (1.7 × 1011 l/mol) and a serum with moderate-affinity IAAs (6.3 × 107 l/mol) (circles). Both curves fit a 2-site binding model. (D) Binding curves obtained for the first IAA-positive serum from 56 children in the BABYDIAB study. IAA binding in 1 serum conforms to a 2-site binding model (dotted line), whereas the remaining sera conform to a 1-site binding model. Curves that are shifted to the right indicate lower-affinity IAAs.
The IAA binding curve observed in the IAA-positive sample was consistent with a 1-site binding model (Figure 1, A and B) and IAAs of high affinity (1.7 × 1011 l/mol). In order to determine whether mixed IAA populations of discrete affinities could be identified by the experimental system, mixing experiments were performed by spiking of the high-affinity IAA-positive serum with serum containing low-affinity (2 × 105 l/mol) or moderate-affinity (6.3 × 107 l/mol) IAAs (Figure 1C). The competitive binding curves of the mixed sera were consistent with a 2-site binding model, and the calculated IAA affinities for each of the IAAs were similar to those determined in the original samples (high-low mix, 1.6 × 1011 l/mol and 2.8 × 105 l/mol; high-moderate mix, 1.2 × 1011 l/mol and 6.2 × 107 l/mol).
IAA competitive binding curves to Tyr14A [125I] insulin were consistent with a 1-site model in the first IAA-positive sample from all but 1 of the 56 children tested, suggesting that IAAs were of relatively homogeneous affinity within each sample (Figure 1D). The IAA binding curve in 1 child aged 9 months was consistent with a 2-site binding model.
IAA affinity varies between children. IAA affinity in the first positive sample from the BABYDIAB children varied substantially between children and ranged from less than 106 l/mol to more than 1011 l/mol (Figure 2). Affinity was not correlated with IAA titer (r = –0.016; P = 0.91) but was associated with HLA DRB1*04 (median affinity, 5.4 × 109 l/mol in HLA DRB1*04 children vs. 9.3 × 108 l/mol in non-DRB1*04 children; P = 0.002) and the age of first IAA detection (P = 0.003, Kruskal-Wallis H test). IAA affinity was greater than 109 l/mol in all 16 children who had IAAs at 9 months (median affinity, 6.8 × 109 l/mol). Only 4 of these 16 children were positive at birth, so the high-affinity IAAs were not due to residual maternal insulin antibodies. The majority (69%) of children in whom IAAs were first detected at age 2 years also had high-affinity IAAs (median affinity, 3.6 × 109 l/mol). In contrast, only 3 of 14 children in whom IAAs were first detected at age 5 or 8 years had affinities above 109 l/mol (median affinity, 2.7 × 108 l/mol; P = 0.004 vs. IAA affinity in children who developed IAAs at age 9 months or 2 years).
Relationship between IAA affinity and the age of IAA appearance or HLA phenotype. (A) IAA affinity of the first IAA-positive sample in children who had the HLA DRB1*04/DQB1*0302 haplotype (HLA DR4) compared with those who did not have this haplotype (non–HLA DR4). (B) IAA affinity of the first IAA-positive sample in BABYDIAB children who developed IAAs at age 9 months or 2 years or at 5 years or older.
IAA affinity is high in children who develop multiple islet autoantibodies. IAA affinity was analyzed with respect to progression to multiple islet autoantibodies and to diabetes (Figure 3A). IAA affinity in the first IAA-positive sample was significantly higher in the 38 children who developed multiple islet autoantibodies (median IAA affinity, 5.4 × 109 l/mol; interquartile range [IQR], 2.7 × 109 to 1.3 × 1010 l/mol) than in the 18 children who did not develop multiple antibodies (median, 5.2 × 107 l/mol; IQR, 1.2 × 107 to 7.0 × 108 l/mol; P < 0.0001). Thirty-six of the 38 children who developed multiple islet autoantibodies and all 20 children who developed T1DM had IAA affinities greater than 109 l/mol, compared with only 2 of 18 of the children who did not progress to multiple islet autoantibodies, including none of 5 who later became IAA negative (transient IAA). IAA affinity in a second group of IAA-positive relatives (Munich family study) was also significantly higher in relatives who had or developed multiple islet autoantibodies (median affinity, 6.9 × 109 l/mol) than in relatives who did not progress to multiple islet autoantibodies (median affinity, 8.1 × 105 l/mol; P = 0.002). Progression to multiple antibodies in both cohorts was not related to IAA titer (Figure 3B).
Relationship between IAA affinity, multiple autoantibodies, and diabetes. (A) IAA affinity (l/mol) in the first IAA-positive sample from 56 children in the BABYDIAB study, in 16 IAA-positive relatives from the Munich family study, and in 11 insulin-treated patients with T1DM. Subjects in the BABYDIAB and Munich family studies are classified as having developed GAD antibodies, IA-2 antibodies, or cytoplasmic islet cell autoantibodies (multiple Ab’s) or not having developed these antibodies (IAAs only). (B) Relationship between IAA affinity (ordinate scale) and IAA titer (abscissa) for the 72 BABYDIAB and Munich family study sera. In A and B, subjects are identified as having developed multiple islet autoantibodies (circles), not having developed multiple islet autoantibodies (crosses), or having transient IAAs (triangles), and as having developed diabetes (filled symbols) or not (open symbols).
In comparison, affinity of insulin antibodies in patients after treatment with subcutaneous insulin was high (median affinity, 2.0 × 109 l/mol) and remarkably consistent between patients (IQR, 1.7 × 109 to 2.2 × 109 l/mol; Figure 3A). In contrast, IAA affinities in 2 sera from blood donors found to be IAA positive in the Diabetes Autoantibody Standardization Program (9) were low (sample M66290, 2.0 × 105 l/mol; sample N05151, 9.2 × 106 l/mol).
High IAA affinity identifies individuals who later progress to multiple islet autoantibodies. Thirty-three of the IAA-positive BABYDIAB children tested did not have other islet autoantibodies in their first IAA-positive sample. In order to determine whether measuring IAA affinity would be useful for distinguishing IAA-positive relatives who would develop multiple islet autoantibodies on follow-up, time-to-event analyses were performed in these 33 children (Figure 4). Progression to multiple islet autoantibodies was 91% within 4 years of follow-up in the 16 children with high-affinity IAAs (>109 l/mol) and was significantly more frequent than in the 17 children with low-affinity IAAs (<109 l/mol; P = 0.0004; Figure 4A). One child with low-affinity IAAs developed multiple islet autoantibodies. Progression to multiple islet autoantibodies in this child was accompanied by a marked increase in IAA affinity. Risk to develop diabetes in the children with high-affinity IAAs was 50% within 6 years (95% confidence interval; 32.1–67.9), whereas none of the children with low-affinity IAAs has developed diabetes (P = 0.02; Figure 4B).
Progression to multiple autoantibodies (A) and diabetes (B) with respect to IAA affinity. Life table analysis of the development of multiple islet autoantibodies was done in 33 BABYDIAB children who were IAA positive without other autoantibodies in their first positive sample. Life table analysis of the development of diabetes was performed for all 56 BABYDIAB children included in the study. Children are categorized as having IAA affinity greater than 109 l/mol (solid line) or less than 109 l/mol (dotted line). Multiple antibodies and diabetes developed more frequently in children with IAA affinity greater than 109 l/mol (P = 0.0004 and P = 0.02, respectively).
IAA affinity is relatively stable during follow-up. The findings indicated that a high-affinity IAA response could occur early in the natural history of T1DM, and that this was predictive of who would develop multiple islet autoantibodies and diabetes. In order to determine whether the lower-affinity responses “matured” and became of higher affinity later in childhood, IAA affinity was determined in 92 follow-up sera from 31 children (Figure 5A). Changes that were greater than 1 log were observed in 4 of these 31 children. IAA affinity increased in follow-up in only 1 of the 11 children with initial IAA affinity less than 109 l/mol. This child was remarkable in that IAAs were positive at age 2 years with an affinity of 108 l/mol, became negative at age 2.7 years, and returned to positive with increased affinity (1.4 × 1011 l/mol) together with GAD antibodies at age 5 years. IAA affinity in a second child increased from 2.7 × 109 l/mol to 7.6 × 1010 l/mol. Two children had decreased IAA affinity on follow-up.
IAA affinity during follow-up. (A) IAA affinity over time (age) for 92 follow-up samples from 31 subjects. Samples are identified as multiple islet autoantibody positive (circles) or IAA positive only (crosses). Samples from individual subjects are connected by lines. IAA affinity increased by more than 1 log in only 2 subjects (thick broken line) and decreased by more than 1 log in 2 subjects (dotted lines). (B) IAA competitive binding curves for consecutive samples from birth in an IAA-positive BABYDIAB child whose sample at age 9 months had binding characteristics consistent with a 2-site binding model. Binding on the ordinate scale is shown as binding (B) relative to maximal binding in the absence of cold insulin (B0). The inset documents IAA titer and affinity at each follow-up visit.
IAA binding curves in follow-up samples from the child with concomitant high- and low-affinity IAAs were informative with respect to affinity maturation (Figure 5B). Samples were available at birth and at approximately 3-month intervals from ages 6 months to 21 months. Insulin antibodies at birth and at 6 months were high affinity (7 × 109 l/mol) without a low-affinity component, consistent with the presence of maternally acquired antibodies to injected insulin. At age 9 months, insulin binding increased markedly. A high-affinity component was present with titer greater than that observed in the 6-month sample, indicating that this included de novo production of IAAs in the child. A predominant low-affinity IAA was also present. Subsequent samples at ages 12, 15, 18, and 21 months had decreasing amounts of both the high- and the low-affinity IAAs until the low-affinity component became undetectable at 18 months. None of the samples from this child contained IgM IAAs (data not shown). This child developed diabetes at age 2.8 years.
Lower-affinity IAAs show a less mature isotype and a restricted IgG subclass distribution. IAA IgG subclasses were measured in the first IAA-positive samples from 44 of the BABYDIAB children. These included 31 with IAA affinity greater than 109 l/mol (30 of whom developed multiple islet autoantibodies) and 13 with IAA affinity less than 109 l/mol. All 31 with high-affinity IAAs had IgG1 IAAs, and 19 of these also had IgG2, IgG3, or IgG4 IAAs. In contrast, 11 of the 13 children with low-affinity IAAs had IAAs consisting of only 1 IgG subclass (P = 0.008 vs. children with high-affinity IAAs), including 1 child with IgG2 IAAs, 1 with IgG3 IAAs, and 1 with IgG4 IAAs (data not shown). The restricted IgG subclass in the lower-affinity IAA samples was independent of IAA titer, which was similar in high- and low-affinity samples.
IgM antibodies are of lower affinity than IgG antibodies. Two children with high-titer low-affinity IAAs (<106 l/mol) had high titers of IgM IAAs, and their IAA binding was abolished when the reaction was performed at room temperature, indicating that these were cold-reactive IgM antibodies to insulin. All the other sera tested had predominantly IgG IAAs, did not have a large IgM component of IAAs, and were reactive both at 4°C and at room temperature (data not shown).
Affinity identifies IAAs with distinct insulin binding characteristics. The wide range of IAA affinity found between subjects suggested that there were substantial differences in the IAA-insulin interaction. We therefore examined binding to alternatively labeled insulin and insulin from different species or insulin analogs. Binding to Tyr19A [125I] insulin was markedly reduced relative to binding to Tyr14A [125I] insulin in some patients (Figure 6). Binding to Tyr19A [125I] insulin was significantly correlated to IAA affinity (r = 0.57; P = 0.001), and IAAs of very low affinity did not bind to Tyr19A [125I] insulin, even when titers against Tyr14A [125I] insulin were high.
[![Relationship between IAA affinity and relative binding to Tyr19A [125I] ins](https://dm5migu4zj3pb.cloudfront.net/manuscripts/21000/21307/small/JCI0421307.f6.gif)
](/articles/view/21307/figure/6)Figure 6
Relationship between IAA affinity and relative binding to Tyr19A [125I] insulin. Percent binding to Tyr19A [125I] insulin relative to binding to Tyr14A [125I] insulin (abscissa) is shown in relation to IAA affinities (ordinate axis) for individual sera. Children who had or developed multiple islet autoantibodies are indicated by circles and those who did not develop multiple islet antibodies by crosses. Filled symbols represent children who developed diabetes.
The affinity-related interference with IAA binding caused by labeling at residue A19 could be explained by steric hindrance of sufficient magnitude to reduce binding of the lower-affinity IAAs, or by IAA epitope differences. In order to determine whether the lower-affinity IAAs were directed against distinct epitopes, we performed competition studies using modified insulin in 65 IAA-positive subjects from the BABYDIAB (n = 54) and Munich family study (n = 11) cohorts (Figure 7, A and B, and Table 1). The majority of subjects (n = 46) had IAAs that bound equally well to human, porcine, and human B28lysB29pro insulin, bound less to sheep A8his insulin and human A13trpB28lysB29pro insulin, and did not bind fish insulin. This pattern corresponded to binding that required conservation of the human sequence within A chain residues 8–10 and 13, but not B chain residues 28–30 (A8–10/13–dependent binding). A second group of subjects (n = 6) had IAAs that bound equally well to all insulins and insulin analogs except fish insulin, which indicated that they were unaffected by changes at A chain residues 8–10 or 13 or B chain residues 28–30 (A8–10/13/B28–30–independent binding). A third group of subjects (n = 13) had IAAs that were affected by changes to insulin B chain residues 28, 29, or 30 (B28–30–dependent binding). These included 3 subjects with IAAs that did not bind to porcine insulin, 3 with IAAs that did not bind to human B28lysB29pro insulin (but bound well to fish insulin), and 7 with IAAs that bound neither human B28lysB29pro nor porcine insulin.
Epitope analysis of IAA. (A) Differences in amino acid sequences in the A and B chains of the insulin molecules used for competition studies of IAA binding. (B) Competitive inhibition of IAA binding to Tyr14A [125I] human insulin using human insulin (open circles), human B28lysB29pro insulin (open triangles), human A13trpB28lysB29pro insulin (shaded squares), porcine insulin (shaded circles), sheep A8his insulin (filled diamonds), and fish insulin (crosses). Five patterns were discernible and are shown by representative sera. Forty-six subjects had IAAs with the A8–10/13–dependent binding pattern represented in the top left panel. Six subjects had IAAs with the A8–10/13/B28–30–independent binding pattern represented in the middle left panel. Three subjects had IAA with the B30-dependent binding pattern represented in the top right panel. Three subjects had the B28/29–dependent binding pattern represented in the middle right panel. Seven subjects had the B28–30–dependent binding pattern represented in the bottom panel. (C) Competitive inhibition of IAA binding to Tyr14A [125I] human insulin using human proinsulin (filled circles) for each of the sera shown in B. The dotted line represents competition with human insulin.
Characteristics of IAA and autoantibody development in IAA-positive relatives
All 44 subjects with high-affinity IAAs had the A8–10/13–dependent binding pattern. In contrast, of the 21 subjects with lower IAA affinity, 13 had IAAs with a B28–30–dependent binding pattern (P < 0.0001 vs. high-affinity IAAs), 6 had IAAs with A8–10/13/B28–30–independent binding (P = 0.0007), and only 2 had IAAs with the A8–10/13–dependent binding pattern (P < 0.0001).
IAA epitope and affinity are related to proinsulin binding. The proinsulin molecule includes a connecting peptide that alters the conformation of COOH-terminal residues of the insulin B chain (10). We therefore asked whether binding to proinsulin could distinguish affinity- and epitope-related IAA reactivity (Figure 7C and Table 1). All B28–B30–independent IAAs, including all high-affinity IAAs, were completely inhibited by both insulin and proinsulin. In marked contrast, all but 1 of the B28–B30–dependent IAAs were poorly inhibited with proinsulin (P < 0.0001). The one exception (case 42b in Table 1) is striking, since this is the low-affinity IAA component in the child with mixed IAA populations. The low-affinity IAAs in this child were uninhibited with porcine insulin and sheep A8his insulin, both of which have residue changes at position B30, but, unlike all other B30-dependent IAAs, were readily inhibited with human proinsulin. The high-affinity IAA component in this child had A8–13–dependent proinsulin binding. Also of note is that the low-affinity IAAs in the first positive sample of the child who subsequently developed multiple islet autoantibodies (case 47) were inhibited with proinsulin. Finally, 2 children had IAAs with affinity less than 109 l/mol with A8–13–dependent and proinsulin binding, and in both cases IAAs were transient (cases 45 and 46).