2. History of Anti-Islet Autoantibody Discovery

Figure 1. Chronology of anti-islet autoantibody discovery. Anti-islet autoantibodies used for prediction and diagnoses of T1D are IAA, GADA, IA-2A, and ZnT8A.

3. Pathophysiology of the Generation of Anti-Islet Autoantibodies

4. Localization and Function of Autoantigens against Anti-Islet Autoantibodies

(1)

Insulin

Insulin is a peptide hormone converted from proinsulin in pancreatic β-cells. Proinsulin consists of an A-chain (21 amino acids), a B-chain (30 amino acids), and a C-peptide (31 amino acids), and mature insulin is generated after C-peptide is excised by a series of proteolytic cleavages. The function of insulin is to regulate glucose levels in the blood and induce glucose storage in the liver, muscles, and adipose tissue.

(2)

GAD 65 and GAD67

GAD is a rate-limiting enzyme that catalyzes a decarboxylation reaction to produce the neurotransmitter γ-aminobutyric acid from L-glutamate. There are two isoforms of GAD, GAD65 (585 amino acids), and GAD67 (594 amino acids), both of which are abundant in the central nervous system and pancreatic islets. In pancreatic β-cells, GAD65 and GAD67 are localized in the synaptic-like vesicles and cytosol, respectively.

(3)

IA-2 and IA-2β/phogrin

IA-2 (also known as ICA512) is a transmembrane glycoprotein of the protein tyrosine phosphatase (PTP) family that colocalizes with IA-2β/phogrin and is expressed in insulin-secretory granule membranes in pancreatic β-cells ^[9][13][9,13]. IA-2 consists of 979 amino acids and IA-2β/phogrin consists of 1015 amino acids. Both polypeptides regulate insulin secretory granule content and pancreatic β-cell growth.

(4)

ZnT8

ZnT8, a member of the zinc transporter family, is specifically localized in the insulin-secretory granule membrane of pancreatic β-cells and consists of 369 amino acids. ZnT8 is responsible for transporting zinc influx from the cytoplasm into insulin granules, facilitating insulin hexamer formation ^[17][18][17,18]. Zinc ions transported into the insulin-secreting granule cavity by ZnT8 are used to stabilize insulin hexamers, and zinc ions ejected outside the cells during insulin secretion have an autocrine effect on β-cells, or alternatively, exert a paracrine action on α-cells.

(5)

Carboxypeptidase H

Carboxypeptidase H is an enzyme which converts proinsulin into insulin and C-peptide by catalyzing the release of C-terminal arginine or lysine residues from polypeptides. This enzyme is localized in the insulin secretory granules and granule membrane of pancreatic β-cells.

(6)

ICA69

Islet-cell autoantigen 69 (ICA69) is localized in the insulin secretory granule membrane and consists of 483 amino acids. This protein is involved in the signaling and maturation of dene-core vesicles.

(7)

GM2-1 ganglioside

GM2-1 ganglioside (N-acetyl neuraminic acid-galactose-galactosamine-galactosamine-glucose-ceramide) is a pancreatic monosialoganglioside migrating between GM2 and GM1 standards which is localized in the secretory granules in β-cells and non-β-cells. However, the function of GM2-1 ganglioside is still unknown.

(8)

Heat shock protein 60

Heat shock proteins were originally described as “cellular stress responders” for their role as chaperones. Heat shock protein 60 (HSP60) consists of 569 amino acids and is localized in the insulin secretory granules. This protein assists in the correct folding of partially folded polypeptides and the presentation of antigen to MHC molecules.

(9)

GLUT2

Glucose transporter-2 (GLUT2) is a subclass of GLUTs and consists of 524 amino acids. GLUT2 is expressed mainly in pancreatic β-cells of the pancreas, liver, and kidney. This transporter is localized on the β-cell surface membrane and is responsible for the transport of glucose from blood into β-cells.

(10)

Tetraspanin-7

Tetraspanin-7 is expressed in the insulin-containing granule membranes of pancreatic β-cells and glucagon-producing α-cells. This protein consists of 249 amino acids and is involved in the regulation of Ca²⁺-dependent insulin exocytosis.

(11)

ICA12/SOX13

ICA12/SOX13 belongs to the class D subgroup of SOX transcription factors and is localized in the cytoplasm and nucleus in β-cells and non-β-cells. ICA12/SOX13 consists of 604 amino acids and contains a leucine zipper domain. The function of this transcription factor in the islets is still unknown.

5. Significance of Anti-Islet Autoantibodies in the Pathophysiology of T1D

The Juvenile Diabetes Research Foundation (JDRF), American Diabetes Association (ADA), and Endocrine Society recommend classifying the natural history of T1D into three stages [27] (Figure 2). In Stage 1, autoimmunity to pancreatic islet cells is induced by cytotoxic T cells, and the destruction of pancreatic β-cells (insulitis) begins, even though blood glucose levels remain within the normal range. Two or more IAA, GADA, IA-2A, and ZnT8A, emerge in this stage, with the five-year and ten-year risks of T1D development being approximately 44% and 70%, respectively, while the lifetime risk approaches 100% [28]. However, it is currently understood that anti-islet autoantibodies do not directly cause pancreatic β-cell destruction; they arise as the result of β-cell destruction mediated by T-cells. In Stage 2, similar to Stage 1, individuals test positive for two or more anti-islet autoantibodies, and β-cell destruction progresses, leading to glucose intolerance or dysglycemia. The five-year risk of T1D development at this stage is approximately 75%, and the lifetime risk approaches 100% [29]. Stage 3 is marked by the manifestations of typical clinical symptoms and signs of T1D, including polyuria, polydipsia, weight loss, general fatigue, diabetic ketoacidosis, and others. In this stage, the amount of residual pancreatic β-cells decreases to 20–30% of normal levels. Since anti-islet autoantibodies appear in the peripheral blood during Stage 1 and Stage 2, they are used as a tool for predicting the onset of T1D. The number of positive autoantibodies is more important for prediction than the specific positive autoantibodies [30].

Figure 2. Three stages of natural history of T1D.

Additionally, a study involving first-degree relatives of patients with T1D reported that IAA or GADA appeared first, followed by IA-2A and ZnT8A directly before the onset of diabetes [31]. We observed a similar sequential emergence of anti-islet autoantibodies in patients with T1D who developed the condition during interferon therapy [32]. Based on this evidence, IA-2A and ZnT8A are considered surrogate markers for pancreatic β-cell destruction. This is supported by a report demonstrating the epitope spreading of IA-2A during the preclinical stage of T1D [33]. Conversely, as shown in Table 2, GADA is detected not only in patients with acute-onset T1D and SPIDDM, but also in some patients with fulminant T1D and non-diabetic patients with polyglandular autoimmune syndrome, autoimmune thyroid disease (AITD), and stiff-person syndrome. Therefore, unlike IA-2A and ZnT8A, it is presumed that GADA may not be a specific marker for pancreatic β-cell destruction. In our case, where GADA became re-elevated more than 10 years after the onset of T1D, anti-thyroid autoantibodies turned positive directly before the re-elevation of GADA [34]. Therefore, it is important to note that GADA may be associated with thyroid autoimmunity rather than insulitis in some instances.

Table 2. Disease specificity of GAD autoantibodies.

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%

LADA, latent-autoimmune diabetes in adults; SPIDDM, slowly progressive insulin-dependent diabetes; OHA, oral hypoglycemic agents.

6. Role of Anti-Islet Autoantibodies in the Diagnosis of T1D

In this section, we discuss the key considerations when diagnosing T1D using anti-islet autoantibodies. First, because distinguishing IAA from insulin antibodies produced by exogenous insulin injection is challenging, we considered patients positive for IAA if antibodies to insulin were present in insulin-naïve patients or those within 2 weeks of initiating insulin therapy. Our study using sera obtained within 2 weeks after onset revealed that the prevalence of IAA, GADA, IA-2A, and ZnT8A in patients with acute-onset T1D was 58, 80, 66, and 63%, respectively (Figure 3A). Similarly, the prevalence of these autoantibodies in SPIDDM patients was 41, 83, 27, and 27%, respectively (Figure 3B). Thus, a combinatorial analysis of the four autoantibodies indicated that 96% of acute-onset T1D and 93% of SPIDDM were immune-mediated T1D, while approximately 5% were classified as idiopathic T1D [35]. These data are consistent with previous reports from studies conducted in countries with Caucasoid populations ^{[10][36][37][38]}[10,36,37,38]. In contrast, the majority of patients with fulminant T1D were negative for anti-islet autoantibodies (Figure 3C) and are classified into idiopathic T1D, suggesting that the mechanism of β-cell destruction in fulminant T1D may differ from acute-onset T1D and SPIDDM.

Figure 3.

Combinatorial analysis of anti-islet autoantibodies in patients with acute-onset T1D (

A

), SPIDDM (

B

), and fulminant T1D (

C

) Adapted with permission from Ref. [35].

In Japan, medical insurance guidelines instruct physicians to measure GADA first when T1D is suspected. However, about 10% of non-fulminant T1D patients are positive for other anti-islet autoantibodies without GADA. Therefore, measuring multiple autoantibodies is crucial for the accurate diagnosis of immune-mediated T1D.

7. Epitopes for Anti-Islet Autoantibodies and Their Clinical Relevance

 

7.1. Insulin Autoantibodies

Fewer epitope analysis studies have been conducted on IAA compared to those of GADA and IA-2A. In an earlier study, the significance of amino acid A13 of the A-chain for the binding of IAA in T1D was demonstrated [39]. Furthermore, it was revealed that conformational epitope spanning amino acid residues A8–A13 on the A-chain and B1–B3 on the B-chain was the major binding site for IAA and was disease-associated. Another epitope analysis using a recombinant Fab of the insulin-specific monoclonal antibody reported that the IAA epitope was located in the A-chain residues A8–A10 [40,41]. However, radioimmunoassay for IAA does not distinguish between epitopes on the insulin molecule bound by IAA from insulin antibodies induced by exogenous insulin injection [38]. Therefore, Devendra and coworkers used the random phage-displayed peptide library and serum obtained from an IAA-positive T1D patient and an insulin-treated insulin autoimmune syndrome patient to examine the difference in epitopes bound by both antibodies [42]. As a result, they identified the two phagotopes (phage that carry peptides that mimic

epitopes), designated IAS-9 and IDD-10, which were able to discriminate between diabetesassociated and non-diabetes associated insulin antibodies. This suggests that phage display technology could potentially be exploited to develop an IAA-specific RIA.

7.2. GAD Autoantibodies

GADA is present in 70–80% of prediabetic relatives and new-onset patients with T1D [30,35]. The major antigenic region of GADA has been determined using truncated peptide or chimeric proteins of GAD65 and GAD67 to maintain the conformational structure, as previous studies reported that GADA in patients with T1D recognizes the conformational structure of the GAD molecule [43]. Moreover, it has been reported that disease-associated GADA is directed against GAD65, and humoral immune reactivity to GAD67 is likely to be cross-reactive to GAD65 [44,45]. Figure 4A shows a schematic representation of the GAD65 and the localization of GADA epitopes in T1D. Previous

studies have demonstrated that GADA in T1D patients recognizes disease-specific GAD65 epitopes, located at the middle and C-terminal regions of GAD65 [46,47]. However, these studies may have also detected epitopes that cross-react with the GAD67 autoantibody. To detect GAD65 autoantibody-specific epitopes, we performed a competitive radioimmunoassay

with recombinant GAD67 protein using a series of GAD65/GAD67 chimeric constructs, and identified the autoantibody epitopes in the N-terminal region (amino acids 1–244; N), the middle domain (amino acids 245–359; E1 and amino acids 360–442; E3), and the C-terminal region (amino acids 443–585; E2) of GAD65 [48]. Although GADA epitope specificities remain relatively stable after the clinical onset of T1D, it has been reported that in genetically predisposed subjects, GADA is initially generated against the middle and C-terminal regions of GAD65. Furthermore, the autoimmune response may undergo intramolecular epitope spreading toward epitopes on the N-terminus and further epitopes located in the middle [49].

7.3. IA-2 Autoantibodies

As shown in Figure 4B, this protein is 979 amino acids long and comprises of a luminal domain (amino acids 27–576), transmembrane domain (amino acids 577–600), and cytoplasmic domain (amino acids 601–979). Although the PTP core sequence is found at amino acids 907–917 in the cytoplasmic domain, the expressed recombinant protein does not exhibit protein tyrosine phosphatase activity. Studies evaluating the biological properties of IA-2 have demonstrated its role as an important regulator of dense core vesicle number as well as glucose-induced and basal insulin secretion [11, 13].

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].

8. Prediction of Future Insulin Deficiency in Patients with SPIDDM (LADA)

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.

9. Conclusions

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

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