Anti-islet autoantibodies serve as key markers in immune-mediated type 1 diabetes (T1D) and slowly progressive T1D (SPIDDM), also known as latent autoimmune diabetes in adults (LADA). Autoantibodies to insulin (IAA), glutamic acid decarboxylase (GADA), tyrosine phosphatase-like protein IA-2 (IA-2A), and zinc transporter 8 (ZnT8A) are currently employed in the diagnosis, pathological analysis, and prediction of T1D. GADA can also be detected in non-diabetic patients with autoimmune diseases other than T1D and may not necessarily reflect insulitis. Conversely, IA-2A and ZnT8A serve as surrogate markers of pancreatic β-cell destruction. A combinatorial analysis of these four anti-islet autoantibodies demonstrated that 93–96% of acute-onset T1D and SPIDDM cases were diagnosed as immune-mediated T1D, while the majority of fulminant T1D cases were autoantibody-negative. Evaluating the epitopes and immunoglobulin subclasses of anti-islet autoantibodies help distinguish between diabetes-associated and non-diabetes-associated autoantibodies and is valuable for predicting future insulin deficiency in SPIDDM (LADA) patients.
Name of Antigen | Localization | Function | Reference |
---|---|---|---|
Insulin | Insulin secretory granules | Regulate glucose levels in the blood and induce glucose storage in the liver, muscles, and adipose tissue | [7] |
GAD65 | Synaptic-like vesicles in the cytoplasm of β-cells | Rate-limiting enzyme engaged in the synthesis of the neurotransmitter γ-aminobutyric acid from L-glutamate | [8] |
GAD67 | Cytosol of β-cells | Rate-limiting enzyme engaged in the synthesis of the neurotransmitter γ-aminobutyric acid from L-glutamate | [11] |
IA-2 | Insulin secretory granule membrane | Regulate insulin secretory granule content and β-cell growth | [9][12][9,12] |
Phogrin/IA-2β | Insulin secretory granule membrane | Regulate insulin secretory granule content and β-cell growth | [13][14][13,14] |
Carboxypeptidase H | Insulin secretory granules and granule membrane | Convert proinsulin into insulin and C-peptide by catalyzing the release of C-terminal arginine or lysine residues from polypeptides | [15] |
ICA69 | Insulin secretory granule membrane | Dense-core vesicles signaling and maturation | [16] |
ZnT8 | Insulin secretory granule membrane | Transport zinc ion from the cytosol into the insulin secretory granules | [17][18][17,18] |
GM2-1 ganglioside | Secretory granules in β-cells and non-β-cells | unknown | [19] |
Heat shock protein 60 | Insulin secretory granules | Assist correct folding of partially folded polypeptides and presentation of antigen to MHC molecules | [20] |
GLUT2 | β-cell surface membrane | Uptake glucose from the blood into β-cells | [21] |
Tetraspanin-7 | Insulin secretory granule membrane | Regulate Ca2+-dependent insulin exocytosis | [22] |
ICA12/SOX13 | Cytoplasm and nucleus in β-cells and non-β-cells | Transcription factor (Function in the islets is unknown) | [23] |
Subject | Prevalence |
---|---|
Healthy control | <1% |
Acute-onset type 1 diabetes (at onset) | 60–80% |
Fulminant type 1 diabetes | 5–9% |
LADA (SPIDDM) | 100% |
Type 2 diabetes (diet/OHA) | 4–5% |
Polyglandular autoimmune syndrome, type 1 | 30–40% |
Polyglandular autoimmune syndrome, type 2 | 30–50% |
Autoimmune thyroid disease | 6–8% |
Stiff-person syndrome | 60–70% |
IA-2A is present in 60-70% of prediabetic relatives and new-onset patients with T1D. We and others have analyzed IA-2A epitopes recognized by diabetic sera using a series of IA-2 fragments or IA-2/phogrin chimeric proteins, and found that the major epitopes are localized in the cytoplasmic domain [51]. Approximately 95% of T1D patients and prediabetic relatives who are IA-2A positive recognize the PTP-like domain (amino acids 687-979), whereas only 5% of sera react with the luminal domain [51, 52]. Furthermore, our binding and competition analysis using multiple IA-2/phogrin chimeric constructs demonstrated that a major unique epitope for IA-2A is localized to amino acids 762-887. A conformational epitope associated with the C-terminal 31 amino acids of IA-2 is recognized by one-third of sera, and a minor epitope is located on amino acids 601-762 of IA-2. Notably, intramolecular epitope spreading was found for relatives of T1D patients who later progressed to T1D. However, relatives who remained nondiabetic exhibited a decrease in the number of recognized epitopes. These studies are consistent with the hypothesis that IA-2 may be recognized as a consequence of β-cell destruction [33].
Another important epitope has been mapped in the juxta-membrane domain of IA-2 (amino acids 601-629; IA-2JM). Our data demonstrated that the age of disease onset in patients with IA-2JMA only was significantly higher than that in patients who reacted with the PTP-like domain, suggesting that autoantibody recognition of IA-2 epitopes in autoimmune diabetes is associated with the age of disease onset, which may reflect the intensity of the β-cell destruction process [53].
7.4. ZnT8 autoantibodies
In 2007, Sladek and coworkers identified four loci containing variants that confer T2D risk through a genome-wide association study, including a non-synonymous polymorphism in the ZnT8 gene (SLC30A8), rs13266634 (C/T), which causes an R325W modification in the protein sequence [54]. In the same year, Hutton and coworkers discovered ZnT8 as a major autoantigen in T1D, and ZnT8A has been recognized as one of the four major anti-islet autoantibodies [23].
ZnT8A are present in 50-60% of prediabetic relatives and new-onset patients with T1D. As shown in Figure 4C, ZnT8 is a 369-amino acid polytopic transmembrane protein with cytoplasmic N- and C- terminal tails. It has been reported that ZnT8A recognizes 101 amino acids localized in the cytoplasmic C-terminal region. In particular, the amino acid residue 325 (R325W) defined by the SLC30A8 polymorphism is critical for humoral autoimmunity to this autoantigen, and binding of ZnT8A against two isotypes (ZnT8-325R, ZnT8-325W) depends on the patient's SLC30A8 genotype [55, 56]. Consequently, heterozygotes with the CT genotype respond to both ZnT8-325R and ZnT8-325W, while CC and TT homozygotes respond exclusively to ZnT8-325R or ZnT8-325W, respectively. Thus, individuals respond to endogenous ZnT8 protein determined by their own genome, and therefore, the current ZnT8A assay, therefore, uses a hybrid protein of two ZnT8 isotypes as antigens.
Furthermore, Wenzlau and coworkers identified that residues 332R, 333E, 336K, and 340K contribute to a conformational ZnT8A epitope independent of residue 325 by comparing human and mouse chimeric ZnT8 proteins [57], suggesting that this epitope may add to the diagnostic utility of measuring ZnT8A.
Figure 4. Illustration of antigenic epitopes recognized by T1D sera in GAD65 (A), IA-2 (B), and ZnT8 (C) proteins.
7.5. Other anti-islet autoantibodies
Other anti-islet autoantibodies include autoantibodies against GM2-1 ganglioside, HSP60, GLUT2, tetraspanin-7, and ICA12/SOX13 (Table 1). Among these, the epitopes of GM2-1 autoantibodies and GLUT2 autoantibodies have not been analyzed so far. It has been reported that HSP60 autoantibodies recognized two epitope regions on HSP60 (amino acids 394-413 and amino acids 435-454). The first region similar to the sequence found in GAD, whereas the second one overlaps with p277 T-cell epitope to a large extent [58]. Using a series of overlapping peptide fragments, Eugster and coworkers mapped map autoepitopes recognized by tetraspsnin-7 autoantibodies and found that autoantibody epitopes lie predominantly within the first and third cytoplasmic domains of the protein. Further characterization of autoantibody binding to mutated constructs revealed that epitopes lie within a relatively short (20-amino acid) region represented by at least two of the three cytoplasmic domains, providing further evidence of the importance of protein conformation in antibody binding [59]. Furthermore, epitope mapping of ICA12/SOX13 autoantibodies using several truncated fragments of SOX13 suggests that autoantibodies are directed to at least two epitopes, one that requires amino acids 66-604, and a second confined within amino acids 327-604 [22].
SPIDDM (also known as LADA) is characterized by the presence of anti-islet autoantibodies and a gradual decline in insulin secretory capacity. The Immunology of Diabetes Society defined LADA as follows; 1) onset of diabetes >35 years, 2) positive test for at least one of the known anti-islet autoantibodies, and 3) requirement of insulin treatment >6 months after the diagnosis of diabetes [60]. LADA encompasses anti-islet autoantibody-positive diabetic patients in both insulin-dependent and non-insulin-dependent states, which is nearly identical to SPIDDM. According to the recently revised diagnostic criteria for SPIDDM, the interval from diabetes diagnosis to the requirement of insulin treatment is >3 months [61]. Additionally, patients with exhausted endogenous insulin secretion (Fasting C-peptide <0.6ng/ml) at the last observed time point are defined as “SPIDDM (definite)”. In contrast, anti-islet autoantibody-positive patients in non-insulin-dependent state are classified as "SPIDDM (probable)" (Table 3). Using this diagnostic criterion, measuring anti-islet autoantibodies other than GADA results in an approximately 3-fold increase in the incidence of SPIDDM among non-insulin-treated diabetic patients compared with measuring GADA alone (2.0-2.4% vs. 7-8%) [62-64]. Indeed, in the Nagasaki Autoimmune Diabetes Intervention/Prevention Study, the prevalence of anti-islet autoantibodies other than GADA in insulin naïve adult-onset diabetes was 8.6%, which is 2.6-fold compared to that of GADA (3.3%) (Figure 5). Since this subtype of T1D is generally indistinguishable from T2D at the time of diagnosis, measuring anti-islet autoantibodies is crucial for early diagnosis and appropriate treatment of SPIDDM (LADA).
Figure 5. Prevalence of GADA, IA-2A, ZnT8A, and IAA in 788 insulin naïve adult-onset patients with diabetes
Anti-islet autoantibody positivity, especially ICA and GADA, is predictive for progression to a future insulin-dependent state after the diagnosis of diabetes. For example, the UKPDS (United Kingdom Prospective Diabetes Study) found that at least 50% of LADA patients required insulin treatment 6 years post-diagnosis [65]. However, not all SPIDDM (LADA) patients required insulin treatment, even after 10 years from diagnosis.
According to a nationwide survey [66] conducted by the Japan Diabetes Society, the predictors of progression to insulin dependent state include (1) age of onset ≤ 47 years, (2) period until GADA positive detection ≤ 5 years, (3) GADA titer (RIA method) ≥ 13.6 U/ml, and (4) fasting C-peptide ≤ 0.65 ng/mL. Additionally, the number of positive anti-islet autoantibodies and GADA epitope recognition are also important for prediction. To identify the predictive markers for early insulin requirement in non-insulin-dependent SPIDDM (probable), we evaluated IAA, IA-2A, and ZnT8A along with GADA-specific epitope recognition in 47 GADA-positive diabetic patients [63]. Among these patients, 38% had one or more of IAA, IA-2A, or ZnT8A and 15% had two or more of these autoantibodies. A high GADA titer (≥ 10U/mL), the presence of GADA-E1, and the presence of one or more among IAA, IA-2A, or ZnT8A at diagnosis marked the risk for early insulin therapy requirement. Furthermore, multiple anti-islet autoantibodies were the most relevant risk factor for the insulin requirement (odds ratio 13.77; 95% CI 2.77-68.45; P<0.001) in a multivariate logistic regression analysis. Therefore, measuring anti-islet autoantibodies other than GADA and ICA is essential for predicting the progression risk of SPIDDM (LADA) patients.
This article focused on reviewing the current understanding of anti-islet autoantibodies in T1D. The clinical utilities of anti-islet autoantibodies in patients with diabetes include diagnosis (immune-mediated or idiopathic), prediction (progressor or non-progressor) and understanding of pathophysiology (insulitis-specific or nonspecific phenomenon) (Figure 6). Since the autoantibody level of anti-islet autoantibodies decreases with disease duration and can become negative, it is essential to measure them early in the onset of T1D for accurate diagnosis. SPIDDM or LADA is often indistinguishable from T2D; therefore, earlier measurement of anti-islet autoantibodies is of great clinical importance for early diagnosis and appropriate treatment. In addition to the anti-islet autoantibody profiles, age of onset and genetic risk score should also be considered for risk triage. Furthermore, the development of a high-throughput assay to detect epitope-specific or immunoglobulin isotype-specific autoantibodies should warrant accurate diagnosis and prediction of autoimmune disorders. Besides, the new type of autoantibody assays, which can simultaneously measure multiple autoantibodies, have the advantages of high sensitivity and specificity, and the ability to measure a large number of samples, making it suitable for large-scale population screening of T1D.
Figure 7. Clinical utilities of anti-islet autoantibodies in patients with diabetes