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Changes in GAD65Ab-Specific Antiidiotypic Antibody Levels Correlate with Changes in C-Peptide Levels and Progression to Islet Cell Autoimmunity E. O¨rtqvist, B. Brooks-Worrell, K. Lynch, J. Radtke, L. M. Bekris, I. Kockum, C.-D. Agardh, C. M. Cilio, A. L. Lethagen, B. Persson, Å. Lernmark, J. Reichow, S. Oak, J. P. Palmer, and C. S. Hampe Departments of Woman and Child Health (E.O¨., B.P.) and Molecular Medicine (I.K.), Karolinska Institutet, SE-171 77 Stockholm, Sweden; Department of Pediatrics (K.L.), Pediatric Epidemiology Center, University of South Florida, Tampa, Florida 33620; Department of Medicine (B.B.-W., J.Ra., L.M.B., S.O., J.P.P., C.S.H.), University of Washington, Seattle, Washington 98109; Department of Clinical Sciences (C.-D.A., C.M.C., A.L.L., Å.L.), Lund University, University Hospital, SE-205 02 Malmo¨, Sweden; Veterans Affairs Puget Sound Health Care System, Department of Medicine (J.Re., J.P.P.), University of Washington, and Seattle Institute for Biomedical and Clinical Research (J.Re.), Veterans Affairs Puget Sound Health Care System, Seattle, Washington 98108

Context: The previously reported absence of 65-kDa glutamate decarboxylase antibody (GAD65Ab)-specific antiidiotypic antibodies (anti-Id) in type 1 diabetes (T1D) patients at clinical onset could be due to an inability to mount an antibody response to GAD65Ab or a longitudinal decline in anti-Id levels. Objective and Design: We investigated anti-Id levels in longitudinal samples obtained from T1D patients (n ⫽ 41) (clinical diagnosis - 12 months), and latent autoimmune diabetes in adults (LADA) patients (n ⫽ 32) who received alum-formulated human recombinant GAD65 (baseline - 12 months). We also determined anti-Id levels in a small cohort of Type 2 diabetes patients during their development of autoimmune T cell responses. Results: At clinical onset T1D patients presented no or low anti-Id levels. However, 22/41 T1D patients showed ⱖ50% increase in GAD65Ab-specific anti-Id levels during follow-up; peaking at 3 (n ⫽ 1), 6 (n ⫽ 10), 9 (n ⫽ 10), or 12 (n ⫽ 1) months. Increasing anti-Id levels marked patients who experienced a temporary increase in C-peptide levels. Anti-Id levels correlated significantly with glycated hemoglobin and C-peptide levels at 6 and 9 months (P values ranged from ⬍0.001 to ⬍0.05). In LADA patients receiving placebo, anti-Id levels declined in seven of nine patients, whereas four of five patients receiving 20 ␮g alum-formulated human recombinant GAD65 showed increasing anti-Id levels. Changes in anti-Id and C-peptide levels closely correlated (P ⬍ 0.0001). The significant decline in anti-Id levels (P ⫽ 0.03) in T2D patients developing T cell autoimmune responses supports our hypothesis that declining anti-Id levels are associated with developing islet autoimmunity. Conclusions: The close association between GAD65Ab-specific anti-Id levels and ␤-cell function may provide a novel marker for the progression of autoimmune diabetes. (J Clin Endocrinol Metab 95: E310 –E318, 2010)

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2010 by The Endocrine Society doi: 10.1210/jc.2010-0785 Received April 4, 2010. Accepted July 6, 2010. First Published Online August 4, 2010

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Abbreviations: Alum-GAD, Alum-formulated human recombinant GAD65; anti-Id, antiidiotypic antibodies; cpm, counts per minute; GAD65, 65-kDa isoform of glutamate decarboxylase; GAD65Ab, GAD65 antibody; IA-2, islet autoantigen 2; HbA1c, glycated hemoglobin; LADA, latent autoimmune diabetes in adults; PAS, protein A-Sepharose; T1D, type 1 diabetes.

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utopsy studies suggest that recent-onset type 1 diabetes (T1D) patients still have about 20% of their ␤-cell mass (1–5). Clinical diagnosis of T1D is often followed by a transient remission period, which is characterized by reduced insulin requirement. This honeymoon period is directly related to residual ␤-cell function at onset (6). Preservation of residual ␤-cell function in patients with autoimmune diabetes is therefore a high-priority focus of diabetes research. Temporary arrest of the autoimmune process in T1D patients with residual ␤-cell function can be induced by immune modulatory therapy with antiCD3 monoclonal antibody (7, 8). In an effort to avoid adverse side effects associated with this treatment (9, 10), antigen-specific immune modulation has been attempted (for review see Ref. 11). Studies in nonobese diabetic (NOD) mice have indicated that administration of the 65kDa isoform of glutamate decarboxylase (GAD65) prevents T1D (for review see Ref. 12). Two studies in small cohorts of individuals with autoimmune diabetes indicated a beneficial effect of injections with alum-formulated human recombinant GAD65 (alum-GAD) on residual C-peptide levels (13, 14). In one of these studies, treatment of patients with latent autoimmune diabetes in adults (LADA) with two consecutive doses of 20 ␮g alum-GAD demonstrated efficacy in preventing ␤-cell destruction that was still significant 5 yr after the injections (15). The underlying mechanisms that are involved in this protection are poorly understood. However, an increase of the CD4⫹CD25⫹/CD4⫹CD25⫺ T cell ratio was observed in the treated LADA patients (13), suggesting an up-regulation of regulatory T cells by alum-GAD treatment. This finding was supported by the recent clinical trial in T1D children (14). In an earlier report, we showed that although alum-GAD treatment of LADA patients induced increased GAD65 antibody (GAD65Ab) levels at higher dosages, GAD65Ab after treatment recognized the same epitopes as before injection, indicating that novel GAD65Ab production had not been initiated by alum-GAD treatment (16). Previously, we demonstrated the presence of GAD65Abspecific antiidiotypic antibodies (anti-Id) in healthy individuals and their relative absence in T1D patients at clinical onset (17, 18). These anti-Id bind the antigen-binding site of the GAD65Ab, preventing thereby the latter’s binding to GAD65. The mechanisms leading to the specific absence of these anti-Id in T1D patients at the time of diagnosis are unclear. In the present study, we investigated the natural course of anti-Id levels in longitudinal samples obtained from T1D patients after clinical onset and the treatment-affected anti-Id levels in LADA patients who received two injections of different dosages of alum-GAD (13). We then confirmed our results in a small cohort of T2D patients developing islet autoimmunity.

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Patients and Methods Patients T1D patients Of the 155 children diagnosed with T1D at St. Go¨ran’s Children’s Hospital or in another pediatric hospital in Stockholm between 1992 and 1995, 129 [median age 8.5 (1.2–16.8) yr; 68 boys] agreed to participate in a prospective study. At the time of diagnosis, before the initiation of insulin therapy, blood samples were drawn for analyses of acid-base balance (base excess), electrolytes, blood glucose, nonfasting C-peptide, glycated hemoglobin (HbA1c), islet cell antibodies, and autoantibodies to GAD65 and islet autoantigen 2 (IA-2). Each patient was hospitalized according to clinical routines at that time for 2– 4 wk (median 20 d). Three weeks after diagnosis, when the patients were in stable metabolic condition and before discharge from the hospital, a standardized breakfast (19) was given to all children from preschool age. Measurements of HbA1c, weight and height, injected insulin dose, GAD65Ab, and IA-2Ab were made at baseline (3 wk) and at every 3 months until 9 or 12 months. Based on sample availability and sample volume, longitudinal samples of 41 children were analyzed for GAD65Ab-specific anti-Id.

LADA patients A detailed description of the participating subjects and the trial design is published elsewhere (13). Briefly, 47 GAD65Abpositive LADA patients (30 –70 yr of age, 39 males) were randomized to one of five groups receiving 4 (n ⫽ 9), 20 (n ⫽ 8), 100 (n ⫽ 9), or 500 (n ⫽ 8) ␮g of alum-GAD or placebo (n ⫽ 13). These patients had been diagnosed with diabetes within the previous 5 yr but had not been treated with insulin. The patients received the first sc injection at wk 1 and an additional injection 4 wk later. Blood samples were taken at baseline and at month 0.25, 1, 2, 6, 9, and 12 after the initial injection. In the present study, 32 patients of the original group were analyzed. The 500-␮g treatment group (n ⫽ 8) was omitted because the significant increase in GAD65Ab titer obscured the analysis of anti-Id. Two patients of the placebo group and the 4-␮g treatment group and three patients of the 100-␮g treatment group were omitted due to lack of complete sample sets (missing C-peptide values).

T2D patients developing islet autoimmunity Samples from adult phenotypic T2D patients (n ⫽ 5) who developed islet autoimmunity were analyzed before (median of 6 months; range, 3–24 months) and after (median of 6 months; range, 3–18 months) development of islet autoimmunity for the presence of GAD65Ab and GAD65Ab-specific anti-Id. Islet autoimmunity was demonstrated by development of T cell responses to islet proteins (as assayed by cellular immunoblotting). None of the patients developed islet cell autoantibodies during the follow-up period.

Plasma C-peptide and HbA1c Fasting plasma C-peptide and C-peptide measurement taken 90 min after a standard breakfast (PCP90) (19) were used as measures of residual C-peptide levels. The C-peptide detection limit in the RIA used (20) was less than 0.1 nmol/liter. All patients measured for fasting plasma C-peptide were also measured for plasma C-peptide 90 min after breakfast. HbA1c was measured with a standard HPLC method, and the upper level for normal was less than 5.3%.

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Autoantibodies GAD65 and IA-2 autoantibodies were determined in radioligand binding assays (21, 22) as standardized in the Third International Combined Autoantibody Workshop (23). Recombinant [35S]GAD65 and [35S]IA-2 were produced by in vitro coupled transcription/translation with SP6 RNA polymerase and nuclease-treated rabbit reticulocyte lysate (Promega, Madison, WI) as previously described (21, 22). The in vitro translated 35 S-labeled antigens were kept at ⫺70 C and used within 2 wk. The serum samples were incubated overnight at 4 C in duplicate with trichloroacetic acid precipitable counts corresponding to 20,000 counts per minute (cpm) of 35S-labeled autoantigen at a final serumdilutionof1:25.Labeledantibody-boundantigenwasseparated from free antigen with protein A-Sepharose (PAS) (Zymed, San Francisco, CA). Bound radioactivity was counted in a 1450 Microbeta (Wallac Oy, Turku, Finland) scintillation counter. The intraassay coefficient of variation was 12% for GAD65Ab. The interassay coefficient of variation for a positive control sample was 15% for GAD65Ab. In the Third International Combined Autoantibody Workshop(23),ourassayshowed70%sensitivityand98%specificity for GAD65Ab and 47% sensitivity and 98% specificity for IA-2Ab. Antibody-positive and -negative samples were included in every assay to correct for interassay variation calculating an autoantibody index [Ab index ⫽ (cpm of tested sample ⫺ cpm of negative standards)/(cpm of positive standard ⫺ cpm of negative standards)] as described (21). The upper limit (index of 0.07 for GAD65Ab and 0.05 for IA-2Ab) of the normal range was established as described (21). The World Health Organization standard (24) was used as the positive standard.

Anti-Id to GAD65Ab The complexes of GAD65Ab and anti-Id in serum samples were dissociated as described earlier (17, 25). Briefly, serum samples (50 ␮l) were incubated with GAD65-specific human monoclonal antibody b96.11 coupled to PAS (50 ␮l of 10% slurry) for 10 min at 55C. After this incubation, the reaction was first incubated at 37 C for 30 min and finally for 10 min at room temperature. The bead volume was titrated for optimal assay conditions. The bound fraction was separated from the unbound serum by centrifugation at 14,000 rpm for 5 min. The supernatant was carefully removed and recentrifuged under the same conditions to assure the absence of b96.11-PAS. The resulting supernatant was analyzed for GAD65Ab by radioligand binding assay. Change in volume was accounted for. Each assay included the World Health Organization standard (24) that was not absorbed to GAD65Ab-PAS and a blank control, which consisted of PBS that had been absorbed to GAD65Ab-PAS. Interassay variation on this sample was approximately 10%. Anti-Id were calculated as the observed increase in GAD65Ab levels after absorption compared with the GAD65Ab level before absorption (index after absorption ⫺ index before absorption) as previously reported (18). Antibody levels were expressed as a relative index to correct for interassay variation using an in-house serum sample obtained from a healthy donor. The blank control verified that b96.11 was not released from the beads during the procedure.

T cell assay: cellular immunoblotting Cellular immunoblotting was performed as previously described (26). Briefly, human islets were subjected to preparative 10% SDS-PAGE, the gels were electroblotted onto nitrocellu-

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lose, nitrocellulose particles were prepared, and the nitrocellulose particles containing islet proteins were used to stimulate peripheral blood mononuclear cells in vitro. Human islets from normal subjects are supplied through the National Institutes of Health Islet Consortium. Positive proliferation was considered to be a stimulation index higher than 2.0, and a designation of islet autoimmunity was given to samples where four to 18 blot sections demonstrated a positive stimulation index (⬎2.0). Excellent discriminant ability for classic T1D with high sensitivity and specificity has been demonstrated for this assay in two masked workshops (27, 28).

Statistical analysis All figures and statistical analysis were drawn or performed using Prism 4. Categorical variables were summarized as frequencies and ␹2 tests tested between groups. When the expected number in cells dropped below five, a Monte Carlo approximation was applied. Correlations were calculated using the Spearman rank correlation test. Median antibody levels between groups were compared using the nonparametric ANOVA (Kruskall-Wallis test) followed by Dunn’s multiple comparisons test. Median antibody levels and C-peptide levels within each group were compared using the Wilcoxon signed rank test. A P value ⬍0.05 was considered significant. National regulatory and local ethics committee approvals were obtained for this study, and written informed consent was obtained from participating individuals or their guardians in accordance with the Declaration of Helsinki.

Results T1D patients at and after clinical onset of disease Longitudinal changes in C-peptide levels During the first year of disease, the T1D patients experienced overall a significant decline in mean (95%) fasting and stimulated log C-peptide levels from ⫺0.69 (⫺0.71 to ⫺0.57) log nmol/liter and ⫺0.45 (⫺0.56 to ⫺0.39) log nmol/ liter, respectively, at baseline to ⫺0.87 (⫺0.9 to ⫺0.8) log nmol/liter (P ⬍ 0.01) and ⫺0.77 (⫺0.87 to ⫺0.67) log nmol/ liter (P ⬍ 0.001), respectively, at 12 months. Forty-eight percent (n ⫽ 20) of the children showed a temporary increase in log fasting C-peptide levels at 6 or 9 months (⫺0.9 to ⫺0.52 log nmol/liter, P ⬍ 0.0001) (data not shown), after which the C-peptide levels declined (data not shown). Likewise, 63% (n ⫽ 26) of the children showed a temporary increase in meal-stimulated log C-peptide levels (⫺0.58 to ⫺0.28 log nmol/liter, P ⬍ 0.0001). Longitudinal changes in anti-Id levels We analyzed longitudinal samples of the T1D patients for the presence of anti-Id specific to GAD65Ab (Fig. 1). We found that at onset, the patients showed no or only low levels of anti-Id (median index 0.057, range 0 – 0.86). However, a 50% or greater increase in GAD65Ab-specific anti-Id levels during follow-up was observed for 22

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Correlation of anti-Id and C-peptide levels Among the 41 patients sampled for anti-Id, the anti-Id levels correlated strongly with levels of C-peptide and HbA1c at 6 and 9 months but not at baseline or 3 months (Table 1). Time plots of mean fasting and stimulated Cpeptide levels showed a delay in the decline of ␤-cell function among patients who showed an increase of at least 50% in anti-Id levels (Fig 2). Patients who experienced the increase in anti-Id levels early (3– 6 months) showed a decline in C-peptide level after 6 months, whereas patients who experienced a later increase in anti-Id levels (9 –12 months) showed no significant decline in C-peptide levels over time. Patients who did not experience an increase in anti-Id levels showed a decline in C-peptide levels immediately after diagnosis.

FIG. 1. GAD65Ab-specific anti-Id levels were analyzed in T1D patients at onset and 3, 6, 9, and 12 months after diagnosis. Anti-Id levels expressed as index for each individual patient are shown. A, Patients showing no increase in anti-Id levels; B, patients showing a significant increase in antiId levels at 3 or 6 months after onset; C, patients showing a significant increase in anti-Id levels at 9 or 12 months after onset.

of 41 patients, peaking at 3 (n ⫽ 1), 6 (n ⫽ 10), 9 (n ⫽ 10), and 12 (n ⫽ 1) months (Fig. 1). This increase was only temporary, and the anti-Id levels decreased subsequently. The remaining 19 patients showed no significant change in their anti-Id level during the analyzed time period.

Alum-GAD-treated LADA patients Samples from the LADA patients enrolled in the alumGAD treatment study were obtained at baseline and at 2, 6, and 9 months and tested for their GAD65Ab titer and GAD65Ab-specific anti-Id. These time points were chosen because all participants had complete datasets with C-peptide levels for these times. There was no correlation between GAD65Ab levels and anti-Id levels (data not shown). In the following, we will emphasize the results obtained from the placebo group and the patient group treated with 20 ␮g alum-GAD, because these two groups had significantly different outcome measures, especially concerning their Cpeptide levels (13). Results obtained from the other dose groups will be shown but not discussed in depth. Anti-Id levels at baseline and after 9 months Anti-Id levels at baseline and after 9 months were determined (Table 2). In the placebo group, nine of 11 patients had detectable anti-Id levels at baseline (median index 0.35, range 0.09 – 0.58). Of these, anti-Id levels declined in seven patients and increased in the remaining two patients.

TABLE 1. Correlation between GAD65Ab-specific anti-Id levels and fasting C-peptide, stimulated C-peptide, HbA1c, and fasting glucose at baseline (3 wk) and at 3, 6, 9, and 12 months after T1D clinical onset Correlation with anti-Id levels (r) Variable Fasting C-peptide (nmol/liter) Stimulated C-peptide (nmol/liter) HbA1c (%) Fasting glucose (nmol/liter) Results are shown as Spearman correlation (r). a

P ⬍ 0.05.

b

P ⬍ 0.01.

c

P ⬍ 0.005.

d

P ⬍ 0.0001.

3 wk ⫺0.24 ⫺0.14 ⫺0.01 ⫺0.14

3 months 0.13 0.13 ⫺0.11 ⫺0.16

6 months 0.34a 0.45c ⫺0.32a ⫺0.00

9 months 0.58d 0.57d ⫺0.44b ⫺0.20

12 months 0.44a 0.52b ⫺0.12 ⫺0.10

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Baseline anti-Id levels did not differ significantly between the two groups (data not shown). Although overall the anti-Id titer in the placebo group declined (mean index, ⫺0.04), an overall increase in anti-Id levels for the 20 ␮g alum-GAD-treated group was observed (median index, 0.14) (P ⫽ 0.02). Summarizing the results from all dose groups (placebo and 4, 20, and 100 ␮g alum-GAD), 18 of 32 patients showed a significant decrease in their anti-Id levels (median index 0.23– 0.05; P ⫽ 0.0003), nine of 32 patients showed a significant increase in their anti-Id levels (median index, 0.1– 0.3; P ⫽ 0.002), two patients showed no change in their anti-Id levels, and five patients had no anti-Id at baseline and did not develop detectable anti-Id during follow-up (Table 2).

FIG. 2. Anti-Id levels in patients experiencing temporary increase in C-peptide level. Fasting C-peptide levels (A), stimulated C-peptide levels (B), and HbA1c (C) are shown at baseline (3 wk), 3, 6, 9, and 9 months after diagnosis for patients whose anti-Id levels increased early (3– 6 months) (䡠 䡠 䡠 䡺 䡠 䡠 䡠), or later (9 –12 months) (- - - f - - -), and for patients whose anti-Id levels did not increase (—x—).

In the patient cohort that received 20 ␮g alum-GAD, five of eight had detectable anti-Id levels at baseline (median index 0.27, range 0.06 – 0.47). Four of these five patients showed an increase in anti-Id levels, whereas the anti-Id level in the remaining patient decreased.

Correlation of changes in anti-Id and fasting C-peptide levels We tested whether the changes in anti-Id levels correlated with changes in fasting C-peptide levels (Table 2 and Fig. 3). In the placebo group, only two of 11 patients showed an increase in fasting C-peptide levels. In these patients, also an increase in anti-Id levels was observed, whereas seven of nine patients with declining fasting C-peptide levels also experienced a decline in anti-Id levels. In the patient cohort that received 20 ␮g alum-GAD, four patients showed an increase in anti-Id levels and Cpeptide levels. One patient’s anti-Id and fasting C-peptide levels decreased. Three patients whose fasting C-peptide levels increased had no detectable anti-Id. Similar correlations were observed in the other two dose groups. Consequently, changes in fasting C-peptide and in anti-Id levels correlated very well in the 32 patients tested (Fig. 3) (P ⬍ 0.0001). Summarizing the results from all four patient groups, all patients (17 of 17) who experienced a decrease in fasting C-peptide levels also showed a decrease in anti-Id levels (Fig. 4A) (P ⫽ 0.0005). Likewise, the majority of patients (70%, nine of 13) who showed an increase in C-peptide levels also showed an increase in anti-Id level (P ⫽ 0.0054) (Fig. 4B).

TABLE 2. Changes in anti-Id levels correlate with changes in fasting C-peptide levels in alum-GAD-treated LADA patients Treatment group Placebo 4 ␮g 20 ␮g 100 ␮g Total NS, Not significant.

n 11 7 8 6 32

Anti-Id increase 2 1 4 2 9

Anti-Id decrease 7 6 1 4 18

C-peptide increase ⴙ anti-Id increase 2 1 4 2 9

C-peptide decrease ⴙ anti-Id decrease 5 5 1 4 17

P value 0.02 NS 0.016 0.016 ⬍0.0001

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FIG. 3. Correlation of changes in fasting C-peptide levels and anti-Id levels in alum-GAD. Changes in fasting C-peptide levels (9 months value ⫺ baseline value) were correlated with changes in anti-Id levels (9 months value ⫺ baseline value). Some values overlap. Linear regression lane is shown.

Noteworthy, of the six patients who developed insulin dependency during the observation time, five had no detectable anti-Id at baseline and also did not develop detectable anti-Id throughout the study. Development of islet cell autoimmunity in T2D is accompanied by a decline in anti-Id to GAD65Ab of the b96.11 specificity To confirm the correlation between the decline in anti-Id and developing islet autoimmunity, phenotypic T2D patients (n ⫽ 5) who were initially negative for islet autoantibodies and T cells responding to human islet antigens and

FIG. 5. GAD65Ab-specific anti-Id decline during development of islet autoimmunity. A, T cell responses of T2D patients before and after they develop islet autoimmunity. Patients’ peripheral blood mononuclear cells were incubated with blot section prepared from human islets. Proliferation of peripheral blood mononuclear cells is expressed as number of positive blot sections. B, Sera of T2D patients who developed islet autoimmunity were analyzed before and after autoimmune development. Anti-Id levels are presented as the difference in GAD65 binding before and after absorption. The ⌬-value is shown as GAD65Ab index. The time relative to development of islet autoimmunity is shown in months. Median binding is indicated.

who subsequently developed T cell autoreactivity to human islet antigens during follow-up (Fig. 5A) were evaluated for the presence of anti-Id. Anti-Id levels were determined before and after development of T cell autoreactivity (Fig. 5B). We found a significant decline in median levels of anti-Id (median index, 0.36 – 0.21; P ⫽ 0.03).

Discussion

FIG. 4. Anti-Id levels at baseline and after 9 months in patients, whose fasting C-peptide levels decreased over time (A) and in patients whose fasting C-peptide levels increased over time (B). Anti-Id levels are presented as the difference in GAD65 binding before and after absorption. The ⌬-value is shown as GAD65Ab index. The time relative to development of islet autoimmunity is shown in months. Median binding is indicated. Some values overlap.

Our previous observation of GAD65Ab-specific anti-Id in healthy individuals and their relative lack in T1D patients at onset has prompted us to investigate the cause of the absence of these antibodies. Absence of anti-Id could be due to an inherent inability to mount an antibody response to GAD65Ab or a longitudinal decline during the prediabetic period. We determined anti-Id titers in two independent longitudinal studies, one representing the natural progression after clinical diagnosis of T1D, the other a clinical study of LADA patients treated with alum-GAD.

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Both studies showed that anti-Id levels can increase and decrease over time, suggesting that anti-Id are part of a dynamic immune response. In a subgroup of T1D patients who experienced a temporary increase in their C-peptide levels after onset, levels of GAD65Ab-specific anti-Id significantly increased 6 or 9 months after diagnosis. T1D has been discussed as a relapsing-remitting disease (29), with the honeymoon period as a last surge of the regulatory immune response before the eventual destruction of the residual ␤-cells. It is therefore of special interest that the temporary increase in anti-Id levels correlated significantly with a delay in the decline of mean C-peptide levels, suggesting a close correlation of disease status and anti-Id levels. A similar close correlation between anti-Id levels and ␤-cell function was also found in the alum-GAD-treated LADA patients. Although the majority of untreated LADA patients showed a decline in anti-Id levels, LADA patients, who received treatment with 20 ␮g alum-GAD showed an increase in anti-Id levels. It is of further interest that five of six patients who developed an insulin requirement during this study lacked anti-Id already at baseline. We suggest that islet autoimmunity in these patients was already further progressed compared with the other patients. In support of this hypothesis, we found that T2D patients who developed T-cell-specific autoimmunity showed a longitudinal decline in their anti-Id levels. Correlations between anti-Id levels and disease status were observed in studies of anti-Id to anti-DNA antibodies in systemic lupus erythematosus, where anti-Id are present in convalescent sera but not in sera taken during active disease (30 –32). Moreover, longitudinal analyses of systemic lupus erythematosus patients showed dynamic changes of anti-Id titers within individuals over a period of time (33). An inverse correlation between anti-Id to autoantibodies that target platelet glycoproteins and disease severity was also reported in patients with idiopathic thrombocytopenic purpura (34). Whether anti-Id have a role in the pathophysiology of autoimmune diseases is unclear. However, we had the opportunity to investigate the anti-Id levels in patients before and after development of islet autoimmunity. Our results point to a direct correlation of the decline in anti-Id levels during the development of islet autoimmunity. Thus, we hypothesize that the decline in anti-Id levels may play a direct role in the development of islet autoimmunity. Anti-Id could exert a regulatory function by neutralizing pathogenic autoantibodies, as has been demonstrated recently, when anti-Id to autoantibodies directed against L-type voltage-gated calcium channels blocked the disruptive effect of these autoantibodies on the autonomic regulation of bladder and colon function in

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mice (35). This finding is in line with earlier observations that anti-Id can inhibit the binding of autoantibodies to their antigens (36, 37). Although no clear pathogenic function of GAD65Ab has been described, GAD65Ab have previously been demonstrated to modulate antigen uptake and presentation (38 – 40). Although the consequences of this modulation are unclear, GAD65Ab could cause the presentation of specific GAD65 peptides to autoreactive T cells and their subsequent stimulation. Neutralization of GAD65Ab by anti-Id could thus interfere with the stimulation of autoreactive T cell responses. Moreover, it has been demonstrated earlier that anti-Id can be taken up by antigen-presenting cells, leading to the presentation of anti-Id-derived peptides to T cells and subsequent T cell stimulation (41). Recently, the uptake of anti-Id by B lymphocytes and presentation of anti-Id peptides on human leukocyte antigen class II molecules, leading to T cell stimulation has been reported (42, 43). These studies suggest an idiotypic network consisting of antibodies, anti-Id, T cells, and B lymphocytes. We speculate that anti-Id either block GAD65Ab, thus preventing the latter from modulating GAD65 uptake and processing, or act as the T-cell-stimulating antigen themselves. Studies aimed at the clarification of this mechanism are currently in progress.

Acknowledgments Address all correspondence and requests for reprints to: Christiane S. Hampe, University of Washington, SLU S-276, 815 Mercer Street, Seattle, Washington 98109. E-mail: [email protected]. The study was performed as independent research sponsored by the National Institutes of Health (DK26190 and DK17047), the Swedish Diabetes Foundation, Swedish Diabetes Association, Swedish Child Diabetes Foundation, the Jerring Foundation, the Foundation of Queen Silvia’s Jubilee Fund for Research on Children and Handicaps, and a Basic Science Award from the American Diabetes Association to (C.S.H.). The study also received support through the Medical Research Service of the Department of Veterans Affairs. Disclosure Summary: The authors have nothing to disclose.

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