Absence of avidity maturation of autoantibodies to the protein tyrosine phosphatase-like IA-2 molecule and glutamic acid decarboxylase (GAD65) during progression to type 1 diabetes

Immunoglobulin G avidity assays are used to distinguish between the acute and chronic phase of several infectious diseases, and there is evidence of autoantibody affinity maturation also in autoimmune diseases. To assess whether the analysis of the avidity of autoantibodies against the protein tyrosine phosphatase-like IA-2 molecule and glutamic acid decarboxylase (GAD65) could improve the accuracy of risk assessment of progression to clinical type 1 diabetes, we established methods for the determination of the autoantibody avidity based on our previously developed time-resolved fluorometric IA-2 and GAD65 autoantibody (IA-2A and GADA) assays. The avidity indices of sequential plasma samples from six IA-2A-positive and seven GADA-positive prediabetic children were analysed applying elution with urea and diethylamine (DEA). For comparison, corresponding avidity indices of control children, who have remained non-diabetic for at least 3 years after seroconversion to IA-2A and GADA positivity, were analysed. For most of the children, only a slight fluctuation in the avidity index values was observed over time, although the titres for IA-2A and GADA varied substantially in some cases. The avidity indices of the prediabetic children remained within the same range as those of the control group throughout the follow-up. Our results indicate that the analysis of the avidity index levels of IA-2A and GADA does not improve the accuracy of the prediction of type 1 diabetes based on autoantibody detection.     

Detection and immunochemical characterization of glutamic acid decarboxylase autoantibodies in patients with autoimmune diabetes mellitus

Since GAD65 undergoes post-translational processing and targeting to subcellular compartments and membranes, it may exhibit different immunochemical properties in the cell context compared with the soluble protein expressed in the cell-free eukaryotic system used in the reference method for GADA assessment (radioligand binding assay (RBA)). In the present work, we detected and characterized GADA in 72 sera from patients with type 1 diabetes mellitus (DM) and 14 sera from adult-onset diabetes patients using analytical systems in which GAD65 is expressed in a cellular context: confocal indirect immunofluorescence (IIF) and electron microscopy after immunogold labeling on monolayers of transfected Chinese hamster ovary (CHO) cells, and immunoprecipitation (IP) of metabolically labeled GAD65. Eighteen serum samples, 16 from type 1 diabetes patients and two from adult-onset diabetes patients, were positive by confocal IIF but scored negative by RBA. All of these 18 sera immunoprecipitated a 65 kDa protein, supporting the existence of the GADA marker in such patients. It may be concluded that GADA negativity by the conventional RBA method using the soluble antigen, as well as negativity for other common markers measured by similar methods, is not enough to rule out the existence of the specific autoimmune component in childhood or adult-onset diabetes. Other analytical methods based in a more physiological presentation of the autoantigen structure, as confocal IIF and IP, bring an extra support to assess the complete repertoire of specific autoantibodies to native-like and membrane-bound, or denatured, beta-cell antigens.     

High-titer autoantibodies against glutamic acid decarboxylase plus autoantibodies against insulin and IA-2 predicts insulin requirement in adult diabetic patients

Antibodies against glutamic acid decarboxylase (GADA) is known to be a good predictive marker for insulin-dependency among adult diabetic patients. However, since not all of the GADA-positive patients will develop insulin requirement, we investigated whether other markers, that is, antibodies against IA-2 (IA-2A), insulin autoantibodies (IAA) and HLA class II type, would affect its predictive value for insulin requirement. Adult diabetic patients in the non-insulin-requiring stage were screened for GADA and registered in the study if positive. At the end of the follow-up period, 15 of the 43 GADA-positive patients required insulin. Among GADA-positive patients, the GADA titers of the insulin-requiring patients were significantly higher (199 U vs. 5.8 U, P<0.001) and high-titer GADA was more frequently detected among insulin-requiring patients (80%vs. 11%, P<0.0001). IAA was more frequently detected in insulin-requiring patients (40%vs. 0%, P<0.001), and IA-2A was detected only among insulin-requiring patients. Combinations of these three antibodies (GADA with either IAA or IA-2A) had 100% positive predictive value. In conclusion, the GADA test is a good screening test for predicting insulin requirement in adult diabetic patients and both the IAA and IA-2A tests are useful second line tests.     

Tyrosine Phosphatase-like Protein (IA-2) and Glutamic Acid Decarboxylase (GAD65) Autoantibodies: A Study of Chinese Patients with Diabetes Mellitus

Type 1 diabetes in most Asian populations may not have a salient autoimmune basis when assessed with single determinations of the major markers, islet cell antibodies (ICAs) and glutamic acid decarboxylase antibodies (GAD65ab). With the inclusion of antibodies to tyrosine phosphatase-like protein IA-2 (IA-2ab) as an additional major marker, we re-examined autoimmune diabetes in a group of Chinese patients. We studied 272 subjects at various stages of disease with blood samples procured for biochemical analysis. ICAs were measured by immunofluorescence, GAD65ab and IA-2ab by radioimmunoassay. Sixty-seven patients fulfilled clinical diagnosis of type 1 diabetes and the remaining 205 patients were type 2. Prevalence of single autoantibody type in recent-onset type 1 diabetes (<1 year duration; n =47 ) showed 10.6% with ICAs, 44.7% GAD65ab and 36.2% IA-2ab. GAD65ab account for more than two-thirds of the markers found in type 1 diabetes. Combined analysis further showed that 51.1% had at least one antibody type, 31.9% with two or more antibodies and 8.5% with all three antibodies. Islet autoimmunity presence in childhood-onset type 1 diabetes improved with the addition of IA-2ab, though less impact was seen in the adult-onset. Similarly, combined analysis for type 2 patients with recent diabetes showed a modest increase to 13% with islet autoimmunity compared to 8% when assessed by GAD65ab alone. Combining IA-2ab and GAD65ab assays results detected slightly more immune-mediated diabetes, compared to using a single GAD65ab determination. Non-autoimmune causes need to be considered in the pathogenesis of type 1 diabetes in Chinese, particularly in adults.     

Programmed death-1 ligands-transfected dendritic cells loaded with glutamic acid decarboxylase 65 (GAD65) inhibit both the alloresponse and the GAD65-reactive lymphocyte response

Type 1 diabetes (T1D) is due to a loss of immune tolerance to islet antigens, such as glutamic acid decarboxylase 65 (GAD65), for which islet transplantation is a promising therapy. Therefore, the generation of tolerance aiming at both alloantigen and GAD65 will help therapeutic intervention greatly in T1D. In this study, we tested the effect of programmed death-1 ligands (PD-L1)-transfected dendritic cells (DC) loaded with GAD65 on the alloresponse and GAD65-reactive lymphocyte response. The DC2.4 cell line was transfected with PD-L1 and co-cultured with GAD65. BALB-c mice were primed, respectively, by intraperitoneal injection with GAD65, PD-L1-transfected- or non-transfected DC (PD-L1/DC or DC), and PD-L1-transfected- or non-transfected DC loaded with GAD65 (PD-L1/DC/GAD65 or DC/GAD65). Splenocytes of treated mice were isolated and restimulated in vitro with GAD65 or the various DC populations above being used as stimulators, respectively. In the mixed lymphocyte reaction, DC/GAD65 were able to stimulate both allogeneic and GAD65-reactive lymphocytes. However, PD-L1/DC/GAD65 were poorer than DC/GAD65 at activating the GAD65-reactive lymphocyte response. Further, although PD-L1/DC could inhibit the alloresponse, PD-L1/DC/GAD65 were more effective at down-regulating the GAD65-reactive lymphocyte response. More importantly, PD-L1/DC/GAD65-primed lymphocytes exhibited the weakest proliferation when again restimulated in vitro by PD-L1/DC/GAD65. Additionally, PD-L1/DC/GAD65 down-regulated interferon-gamma and up-regulated interleukin-10 production by activated lymphocytes. Therefore, combined stimulation in vivo and in vitro by PD-L1/DC/GAD65 could inhibit both the alloresponse and the GAD65-reactive lymphocyte response, which may contribute to controlling diabetes and islet transplant rejection.     

Antibody cross-reactivity induced by the homologous regions in glutamic acid decarboxylase (GAD65) and 2C protein of coxsackievirus B4

GAD₆₅ contains an amino acid sequence which is highly homologous with a sequence in the 2C protein of coxsackievirus B4 (CBV4-2C). In the present study the possibility that this region could contain an epitope capable of inducing immunological cross-reactivity between CBV4-2C and GAD₆₅ was evaluated. Six out of seven rabbit sera, which were raised against seven different synthetic peptides carrying various modifications of the homology sequence, showed cross-reactivity between 2C, GAD₆₅ and GAD₆₇ derived peptides in ELISA. There was substantial cross-reactivity between 2C and GAD₆₅ peptides, but not between 2C and GAD₆₇ peptides. The most cross-reactive peptides were those corresponding to the 2C sequences FIEWLKVKILPEVKEK and KILPEVKEKHEFLSRL. When the binding of the four 2C peptide-specific sera to the GAD₆₅ protein was analysed in immunoprecipitation, two sera were found to be cross-reactive (anti-FIEWLKVKILPEVKEK and anti-WLKVKILPEVKEKHEF). One of these (anti-WLKVKILPEVKEKHEF) reacted also with coxsackie B virus (CBV)-infected cells. Antibodies against this epitope were induced during enterovirus (including CBV) infections in initially healthy children who later progressed to clinical insulin-dependent diabetes mellitus (IDDM). Antibody responses were frequent also in constantly GAD₆₅ antibody-negative non-diabetic children, and antibody levels did not differ between newly diagnosed IDDM patients and matched control subjects. Blocking experiments confirmed that the observed reactivity of both rabbit and human antibodies was immunologically specific. The results suggest that the epitope is antigenically highly similar in 2C and GAD₆₅, and that peptide immunization induces antibodies which cross-react with these molecules. However, the significance of this phenomenon in the pathogenesis of IDDM remains to be confirmed, as the peptide antibody levels were similar in patients with recent-onset IDDM and in control subjects.     

Comparative analysis of epitope recognition of glutamic acid decarboxylase (GAD) by autoantibodies from different autoimmune disorders

Autoantibodies to GAD, an important marker of the autoimmune process in type I or insulin-dependent diabetes mellitus (IDDM), are also found in non-diabetic individuals with autoimmune polyendocrine syndrome type 1 (APS1), APS2, and stiff man syndrome (SMS). Most IDDM sera contain two distinct GAD antibody specificities, one of which targets an epitope region in the middle-third of GAD65 (IDDM-E1; amino acids 221-359) and one of which targets the carboxy-third of GAD65 (IDDM-E2; amino acids 453-569). Using 11 chimeric GAD65/GAD67 proteins to maintain conformation-dependent epitopes of GAD65, we compared the humoral repertoire of IgG antibodies from an individual with APS2-like disease (b35, b78, and b96) and MoAbs from an IDDM patient (MICA-2, MICA-3, and MICA-4). Neither the APS2 IgG antibodies nor the IDDM MoAbs bind the amino-terminal third of GAD65, but instead target the carboxy-terminal two-thirds of GAD65. Amino acids 270-359 (IDDM-E1) are targeted by one APS2 IgG antibody and MICA-4, while two other APS2 IgG antibodies, MICA-2 and MICA-3, target amino acids 443-585 (IDDM-E2). Using GAD65/67 chimera that span the IDDM-E2 region, we found that MICA-2 binds amino acids 514-528 of GAD65, but two APS2 IgG antibodies require this region and amino acids 529-570. In contrast, the binding of MICA-3 requires two discontinuous amino acid segments of GAD65 (452-513 and 528-569), but not amino acids 514-528. These results indicate that there are both similarities and differences in the humoral response to GAD65 in APS2 and IDDM.     

Quantitative analysis of islet glutamic acid decarboxylase p64 autoantibodies in insulin-dependent diabetes mellitus.

Autoantibodies against the beta-cell M(r) 64,000 protein (p64), recently identified as an isoform of glutamic acid decarboxylase (GAD), are prevalent in patients with insulin-dependent diabetes mellitus (IDDM). Dog islets were found to represent an abundant source of native p64 allowing the study of antigen-antibody interactions in IDDM. A quantitative, standardized assay for p64 antibodies based on dog islets was developed and evaluated. Utilizing dog and human islets the p64 antibodies were detected in 17/19 (89%) new onset 15-32-year-old patients, compared to 15/19 (79%) in a rat islet assay. ICA were detected in 15/19 (79%) patients and correlated with the presence of p64 antibodies (rs = 0.59, P < 0.004) but not with age at onset, sex, or C-peptide levels. Sensitivity therefore is improved with the dog islet p64 antibody assay which will allow future studies requiring native p64 antigen in larger quantities are possible based on our findings.     

Only multiple autoantibodies to islet cells (ICA), insulin, GAD65, IA-2 and IA-2beta predict immune-mediated (Type 1) diabetes in relatives.

We report here our prospective study of 15,224 non-diabetic, first-degree relatives of probands with immune-mediated (type 1) diabetes (IMD), of which 135 were found to eventually develop diabetes. We determined islet cell, insulin, GAD65, insulinoma-associated antigen-2 and 2beta autoantibodies (ICA, IAA, GAD65A, IA-2A and IA-2betaA), on the first available serum samples. The latter three autoantibodies were however assayed on subsets of the relatives with and without ICA, IAA and/or GAD65A, plus most of the relatives who developed diabetes. Of the relatives who progressed to diabetes, 94% had at least one of these autoantibodies on the first screening, while ICA proved to be the most sensitive single marker (sensitivity 74%). Risk of diabetes was however negligible when ICA was found in the absence of the others (5-year risk=5.3%), but increased dramatically whenever two or more autoantibodies were present (5-year risk=28.2% and 66.2%, respectively). The most predictive combination of markers was ICA plus IA-2A and/or IA-2beta A. Loss of first phase insulin release to IVGTT also occurred only in those ICA-positive relatives who had one or more of the other autoantibodies. The data suggests that significant beta-cell damage is seen only when the underlying autoimmunity has spread to multiple antigenic islet cell determinants. Combinations of the autoantibodies occurred most often in relatives with the highest risk HLA-DR/DQ phenotypes. These data document that only relatives positive for at least two or more of these five autoantibodies are at significant risk of diabetes themselves. Intervention trials for the prevention of type 1 diabetes could be designed based on testing for these autoantibodies alone, without the need for HLA typing and IVGTT testing.     

Longitudinal changes in epitope recognition of autoantibodies against glutamate decarboxylase 65 (GAD65Ab) in prediabetic adults developing diabetes

We analysed the beta cell-specific autoimmunity reflected in autoantibodies to the smaller isoform of glutamate decarboxylase (GAD65Ab) in the prediabetic period of GAD65Ab-positive healthy adults who developed Type 2 diabetes (T2D) during a follow-up period of 10 years. We found that of the adults that tested GAD65Ab-positive at baseline (n=25), six developed T2D and one developed Type 1 diabetes (T1D). Of the subjects that tested GAD65Ab-negative at baseline (n=2209), 81 developed T2D, one developed T1D and four developed unclassified diabetes, indicating that the risk for GAD65Ab-positive healthy adults to develop diabetes is increased sixfold. The GAD65Ab epitopes were characterized in a competition radioligand binding assay using recombinant Fab derived of GAD65-specific monoclonal antibodies. We observed that the GAD65Ab epitope specificities in the prediabetic period changed dynamically. Specifically, the binding to a middle and a C-terminal epitope increased during the follow-up period (P=0 x 03), causing a significant increase in the number of epitopes recognized (P=0 x 03). These findings are similar to previous observations of dynamic changes in the prediabetic period of schoolchildren at high risk for T1D development. However, the character of the epitopes differs between the two populations, suggesting differences in the beta cell-specific autoimmune response in the prediabetic period of patients with latent autoimmune diabetes in adults (LADA) and T1D.     

Progression to type 1 diabetes is associated with a change in the immunoglobulin isotype profile of autoantibodies to glutamic acid decarboxylase (GAD65). Childhood Diabetes in Finland Study Group

To investigate whether type 1 diabetes in man is associated with a preferential Th1/Th2 response, and whether autoantibodies to one of the main autoantigens would reflect such a response, we characterized the immunoglobulin isotype profile to the 65-kDa isoform of glutamic acid decarboxylase (GAD65) in siblings to IDDM patients. Samples obtained from affected subjects before and at clinical onset of IDDM, from unaffected individuals at high risk and at low risk and from healthy controls were studied. The immunoglobulin isotype profile in the siblings at low risk reflected a more immature, i.e., IgM and Th2 like, i.e., IgE response compared to the progressors and siblings at high risk, with significantly higher median levels of IgM and IgE. The rank order of anti-GAD65 immunoglobulin isotypes was similar in the siblings before and at clinical onset of IDDM, IgG1 > IgG4 > IgM > IgE > IgA > IgG3 > IgG2, but markedly different in the individuals at low risk, IgG1 > IgM > IgE > IgG4 > IgG3 > IgA > IgG2. Based on these observations, we suggest that progression to clinical onset of IDDM is associated with a maturation and a decrease in the Th2 immune response against GAD65; findings which could have implications for future intervention and prediction strategies.     

Engineered variants of human glutamic acid decarboxylase (GAD) and autoantibody epitope recognition

Of the two homologous forms of glutamic acid decarboxylase, GAD65 and GAD67, only GAD65 is a common target of autoimmunity. Epitope profiles of autoantibodies to GAD65 (GADA) in 140 type 1 diabetes, adult-onset diabetes mellitus (AODM), and thyroid diseases (TD) were studied. Probes were GAD65, GAD65/67 hybrids (displaying separately GAD65 residues 1-95, 96-444, and 445-585), delta GAD65 (a truncated GAD65 spanning residues 69-585), and GAD67. delta GAD65 and GAD65 detected 137 and 125 positive patients, respectively. The hybrids reacted with 113 sera and in 3 cases disclosed cryptic epitopes. Eighteen patients reacted with GAD67, indicating GAD65-GAD67 cross-reactivity. Most patients recognized both middle and C-terminal epitopes, had low reactivity against N-terminal epitopes, and seldom displayed reactivity limited to the N or C terminus. Compared with type 1 and AODM, TD patients showed a greater prevalence of multiple reactivity and higher incidence of GAD67 positivity.     

Severe anaphylactic reactions to glutamic acid decarboxylase (GAD) self peptides in NOD mice that spontaneously develop autoimmune type 1 diabetes mellitus

Background

Insulin dependent (i.e., "type 1") diabetes mellitus (T1DM) is considered to be a T cell mediated disease in which T_H1 and T_c autoreactive cells attack the pancreatic islets. Among the beta-cell antigens implicated in T1DM, glutamic acid decarboxylase (GAD) 65 appears to play a key role in the development of T1DM in humans as well as in non-obese diabetic (NOD) mice, the experimental model for this disease. It has been shown that shifting the immune response to this antigen from T_H1 towards T_H2, via the administration of GAD65 peptides to young NOD mice, can suppress the progression to overt T1DM. Accordingly, various protocols of "peptide immunotherapy" of T1DM are under investigation. However, in mice with experimental autoimmune encephalomyelitis (EAE), another autoimmune T_H1 mediated disease that mimics human multiple sclerosis, anaphylactic shock can occur when the mice are challenged with certain myelin self peptides that initially were administered with adjuvant to induce the disease.

Results

Here we show that NOD mice, that spontaneously develop T1DM, can develop fatal anaphylactic reactions upon challenge with preparations of immunodominant GAD65 self peptides after immunization with these peptides to modify the development of T1DM.

Conclusions

These findings document severe anaphylaxis to self peptide preparations used in an attempt to devise immunotherapy for a spontaneous autoimmune disease. Taken together with the findings in EAE, these results suggest that peptide therapies designed to induce a T_H1 to T_H2 shift carry a risk for the development of anaphylactic reactivity to the therapeutic peptides.

     

T cell response pattern to glutamic acid decarboxylase 65 (GAD65) peptides of newly diagnosed type 1 diabetic patients sharing susceptible HLA haplotypes.

Autoantibodies and autoreactive T lymphocytes directed against several pancreatic beta cell proteins such as GAD65 have been identified in the circulation before and at the onset of clinical type 1 (insulin-dependent) diabetes. Using GAD65 synthetic peptides, we studied the proliferative response of peripheral blood mononuclear cells (PBMC) either from recently diagnosed type 1 diabetic patients, of whom the majority share the disease-associated HLA class II haplotype (DR4-DQB1*0201 or DR3-DQB1*0302), or from HLA-matched control subjects. We found that 67% (14/21) of the type 1 diabetic patients and 39% (9/23) of the control subjects exhibited a positive proliferative response. Compared with control subjects, however, PBMC from diabetic patients proliferated more frequently (P < 0.05) in the presence of peptide pools from the C-terminal region of GAD65 (amino acids 379-585). Diabetic patients with the same HLA-DQ or HLA-DR alleles showed partially identical T cell reactivity, but no clear correlation could be made between MHC class II specificity and T cell epitopes because of multiple combinations of class II alleles. In addition, by flow cytometry, we studied the direct binding of GAD65 peptides to MHC class II molecules of Epstein-Barr virus (EBV)-transformed B (EBV-B) cells obtained from a diabetic patient. We found that 11 GAD peptides were able to bind to the highly susceptible haplotype DRB1*0301/0401-DQA1*0301/0501-DQB1*0302/0201 on the surface of EBV-B cells in partial correlation with the results obtained in the proliferation assays.     

Autoantibodies to glutamic acid decarboxylase (GAD) detected by an immuno-trapping enzyme activity assay: relation to insulin-dependent diabetes mellitus and islet cell antibodies.

It has recently been proposed that the islet 64,000 Mr protein autoantigen (64K) of insulin-dependent diabetes mellitus (IDDM) is glutamic acid decarboxylase (GAD). We evaluated, by means of a newly developed immunotrapping enzyme activity assay (ITEAA), the prevalence of circulating GAD-autoantibodies (Ab) in a large population of IDDM patients (n = 168), blood donors (n = 87) and non-diabetic autoimmune patients (n = 40). The latter two groups were used as controls. Overall, GAD-Ab were found in 22% of IDDM patients, but in none of the two control groups (P = 0.007). These specificities were invariably associated with islet cell antibodies (ICA) (31.6% in IDDM with ICA vs 0 in IDDM without ICA, P = 0.0001), and this prevalence was higher in sera with high titer ICA (54.5% in IDDM with ICA greater than 80 JDF-units vs 22.6% of IDDM with ICA 5-80 JDF units; P = 0.002). Moreover, GAD-Ab were associated with the female sex (P = 0.002) and the concomitant presence of thyroid and/or gastric antibodies (P = 0.002). No correlation was observed between GAD-Ab and age of the patients, duration of IDDM, or associated non-organ specific antibodies. Our study indicates that GAD-Ab measured by ITEAA are: (1) detected in a proportion of IDDM patients; (2) strongly associated with ICA; (3) preferentially found in IDDM female patients with autoimmune polyendocrine serology; and (4) detected with lower frequency than that reported for 64K-Ab in IDDM.     

Comparison through immunoblotting of glutamic acid decarboxylase (GAD) from newborn and adult rat brain

Immunochemical characterization of glutamic acid decarboxylase (GAD) from brain extracts of newborn and adult rats was investigated using a GAD antiserum that was previously raised against brain GAD from adult rats. According to the immunoprecipitation and saturation curves, no significant differences could be found as to the recognition of newborn and adult GAD by the antiserum. On immunoblots, both extracts revealed the same two immunolabelled bands (mol.wt. 59,000 and 62,000 ± 2000 Da). In both cases, the lightest band showed the strongest staining. Quantitative analysis of the immunolabelling indicated that each immunolabelled band was enriched about 10-fold in the adult brain extract. These data did not reveal any difference between newborn and adult GAD that was reminiscent of the difference found in an earlier study between GAD from lower and higher vertebrates. Whatever the regulatory mechanism responsible for the presence of two forms of GAD in the adult brain, it is already fully operative in newborn animals.     

Glutamic acid decarboxylase 65 and islet cell antigen 512/IA-2 autoantibodies in relation to human leukocyte antigen class II DR and DQ alleles and haplotypes in type 1 diabetes mellitus

The frequencies of autoantibodies against glutamic acid decarboxylase 65 (GAD65) and islet cell antigen (ICA) 512/IA-2 (512/IA-2) are functions of the specific human leukocyte antigen (HLA) in type 1 diabetes mellitus (T1D). We investigated the association of HLA class II (DR and DQ) alleles and haplotypes with the presence of GAD and IA-2 autoantibodies in T1D. Autoantibodies were tested in 88 Tunisian T1D patients and 112 age- and gender-matched normoglycemic control subjects by enzyme immunoassay. Among T1D patients, mean anti-GAD antibody titers were higher in the DRB1*030101 allele (P < 0.001), together with the DRB1*030101/DQB1*0201 (P < 0.001) and DRB1*040101/DQB1*0302 (P = 0.002) haplotypes, while lower anti-GAD titers were associated with the DRB1*070101 (P = 0.001) and DRB1*110101 (P < 0.001) alleles and DRB1*070101/DQB1*0201 (P = 0.001) and DRB1*110101/DQB1*030101 (P = 0.001) haplotypes. Mean anti-IA-2 antibody titers were higher in the DRB1*040101 allele (P = 0.007) and DRB1*040101/DQB1*0302 (P = 0.001) haplotypes but were lower in the DRB1*110101 allele (P = 0.010) and the DRB1*110101 (P < 0.001) and DRB1*110101/DQB1*030101 (P = 0.025) haplotypes. Multinomial regression analysis confirmed the positive association of DRB1*030101 and the negative association of DRB1*110101 and DQB1*030101, along with the DRB1*070101/DQB1*0201 and DRB1*110101/DQB1*030101 haplotypes, with anti-GAD levels. In contrast, only the DRB1*040101/DQB1*0302 haplotype was positively associated with altered anti-IA-2 titers. Increased GAD65 and IA-2 antibody positivity is differentially associated with select HLA class II alleles and haplotypes, confirming the heterogeneous nature of T1D.