Mikael Chéramy

GAD Treatment and Insulin Secretion in Recent-Onset Type 1 Diabetes

BACKGROUND The 65-kD isoform of glutamic acid decarboxylase (GAD) is a major autoantigen in patients with type 1 diabetes mellitus. This trial assessed the ability of alum-formulated GAD (GAD-alum) to reverse recent-onset type 1 diabetes in patients 10 to 18 years of age. METHODS We randomly assigned 70 patients with type 1 diabetes who had fasting C-peptide levels above 0.1 nmol per liter (0.3 ng per milliliter) and GAD autoantibodies, recruited within 18 months after receiving the diagnosis of diabetes, to receive subcutaneous injections of 20 microg of GAD-alum (35 patients) or placebo (alum alone, 35 patients) on study days 1 and 30. At day 1 and months 3, 9, 15, 21, and 30, patients underwent a mixed-meal tolerance test to stimulate residual insulin secretion (measured as the C-peptide level). The effect of GAD-alum on the immune system was also studied. RESULTS Insulin secretion gradually decreased in both study groups. The study treatment had no significant effect on change in fasting C-peptide level after 15 months (the primary end point). Fasting C-peptide levels declined from baseline levels significantly less over 30 months in the GAD-alum group than in the placebo group (-0.21 vs. -0.27 nmol per liter [-0.62 vs. -0.81 ng per milliliter], P=0.045), as did stimulated secretion measured as the area under the curve (-0.72 vs. -1.02 nmol per liter per 2 hours [-2.20 vs. -3.08 ng per milliliter per 2 hours], P=0.04). No protective effect was seen in patients treated 6 months or more after receiving the diagnosis. Adverse events appeared to be mild and similar in frequency between the two groups. The GAD-alum treatment induced a GAD-specific immune response. CONCLUSIONS GAD-alum may contribute to the preservation of residual insulin secretion in patients with recent-onset type 1 diabetes, although it did not change the insulin requirement. (ClinicalTrials.gov number, NCT00435981.)

GAD-alum treatment in patients with type 1 diabetes and the subsequent effect on GADA IgG subclass distribution, GAD65 enzyme activity and humoral response

We have previously shown that two injections of 20 μg GAD-alum to recent onset type 1 diabetic children induced GADA levels in parallel to preservation of insulin secretion. Here we investigated if boosted GADA induced changes in IgG1, 2, 3 and 4 subclass distributions or affected GAD(65) enzyme activity. We further studied the specific effect of GAD-alum through analyses of IA-2A, tetanus toxoid and total IgE antibodies. Serum from children receiving GAD-alum or placebo was collected pre-treatment and after 3, 9, 15 and 21 months. At 3 months a reduced percentage of IgG1 and increased IgG3/IgG4 were detected in GAD-alum treated. Further, IA-2A, IgE and tetanus toxoid antibodies, as well as GAD(65) enzyme activity, were unaffected confirming the specific effect of treatment. In the GAD-alum group, higher pre-treatment GADA were associated to more pronounced C-peptide preservation. The induced IgG3/IgG4 and reduced IgG1 suggest a Th2 deviation of the immune response.

Long-Lasting Immune Responses 4 Years after GAD-Alum Treatment in Children with Type 1 Diabetes

A phase II clinical trial with glutamic acid decarboxylase (GAD) 65 formulated with aluminium hydroxide (GAD-alum) has shown efficacy in preserving residual insulin secretion in children and adolescents with recent-onset type 1 diabetes (T1D). We have performed a 4-year follow-up study of 59 of the original 70 patients to investigate long-term cellular and humoral immune responses after GAD-alum-treatment. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with GAD65. Frequencies of naïve, central and effector memory CD4+ and CD8+ T cells were measured, together with cytokine secretion, proliferation, gene expression and serum GAD65 autoantibody (GADA) levels. We here show that GAD-alum-treated patients display increased memory T-cell frequencies and prompt T-cell activation upon in vitro stimulation with GAD65, but not with control antigens, compared with placebo subjects. GAD65-induced T-cell activation was accompanied by secretion of T helper (Th) 1, Th2 and T regulatory cytokines and by induction of T-cell inhibitory pathways. Moreover, post-treatment serum GADA titres remained persistently increased in the GAD-alum arm, but did not inhibit GAD65 enzymatic activity. In conclusion, memory T- and B-cell responses persist 4 years after GAD-alum-treatment. In parallel to a GAD65-induced T-cell activation, our results show induction of T-cell inhibitory pathways important for regulating the GAD65 immunity.

GAD‐treatment of children and adolescents with recent‐onset type 1 diabetes preserves residual insulin secretion after 30 months

This study aimed to analyse data from two different studies (phase II and phase III) regarding the safety and efficacy of treatment with alum formulated glutamic acid decarboxylase GAD65 (GAD‐alum) at 30 months after administration to children and adolescents with type 1 diabetes.

Cellular and Humoral Immune Responses in Type 1 Diabetic Patients Participating in a Phase III GAD-alum Intervention Trial

OBJECTIVE GAD formulated in aluminum hydroxide (GAD-alum) has previously been shown to induce preservation of residual insulin secretion in recent-onset type 1 diabetes, but recent phase II and III GAD-alum trials failed to reach primary outcomes. The European phase III study was therefore closed after 15 months, and only a minority of patients completed the 30 months of follow-up. RESEARCH DESIGN AND METHODS This study aimed to characterize cellular and humoral responses in the Swedish patients (n = 148) participating in the phase III trial, receiving four (4D) or two (2D) GAD-alum doses or placebo. Serum GAD65 antibody (GADA) levels, GADA IgG1–4 subclass distribution, cytokine secretion, and proliferative responses in peripheral blood mononuclear cells (PBMCs) were analyzed. RESULTS The GAD65-induced cytokine profile tended to switch toward a predominant Th2-associated profile over time both in the 2D and 4D group. The groups also displayed increased GADA levels and PBMC proliferation compared with placebo, whereas GADA IgG subclass distribution changed in 4D patients. CONCLUSIONS Both 2D and 4D patients displayed GAD65-specifc cellular and humoral effects after GAD-alum treatment, but at different time points and magnitudes. No specific immune markers could be associated with treatment efficacy.

Regulatory T cell phenotype and function 4 years after GAD–alum treatment in children with type 1 diabetes

Glutamic acid decarboxylase (GAD)65 formulated with aluminium hydroxide (GAD‐alum) was effective in preserving insulin secretion in a Phase II clinical trial in children and adolescents with recent‐onset type 1 diabetes. In addition, GAD‐alum treated patients increased CD4+CD25hi forkhead box protein 3+ (FoxP3+) cell numbers in response to in‐vitro GAD65 stimulation. We have carried out a 4‐year follow‐up study of 59 of the original 70 patients to investigate long‐term effects on the frequency and function of regulatory T cells after GAD‐alum treatment. Peripheral blood mononuclear cells were stimulated in vitro with GAD65 for 7 days and expression of regulatory T cell markers was measured by flow cytometry. Regulatory T cells (CD4+CD25hiCD127lo) and effector T cells (CD4+CD25–CD127+) were further sorted, expanded and used in suppression assays to assess regulatory T cell function after GAD‐alum treatment. GAD‐alum‐treated patients displayed higher frequencies of in‐vitro GAD65‐induced CD4+CD25+CD127+ as well as CD4+CD25hiCD127lo and CD4+FoxP3+ cells compared to placebo. Moreover, GAD65 stimulation induced a population of CD4hi cells consisting mainly of CD25+CD127+, which was specific of GAD‐alum‐treated patients (16 of 25 versus one of 25 in placebo). Assessment of suppressive function in expanded regulatory T cells revealed no difference between GAD‐alum‐ and placebo‐treated individuals. Regulatory T cell frequency did not correlate with C‐peptide secretion throughout the study. In conclusion, GAD‐alum treatment induced both GAD65‐reactive CD25+CD127+ and CD25hiCD127lo cells, but no difference in regulatory T cell function 4 years after GAD‐alum treatment.

Characteristics of in‐vitro phenotypes of glutamic acid decarboxylase 65 autoantibodies in high‐titre individuals

Previous studies have indicated phenotypical differences in glutamic acid decarboxylase 65 autoantibodies (GADA) found in type 1 diabetes (T1D) patients, individuals at risk of developing T1D and stiff‐person syndrome (SPS) patients. In a Phase II trial using aluminium‐formulated GAD65 (GAD‐alum) as an immunomodulator in T1D, several patients responded with high GADA titres after treatment, raising concerns as to whether GAD‐alum could induce GADA with SPS‐associated phenotypes. This study aimed to analyse GADA levels, immunoglobulin (Ig)G1–4 subclass frequencies, b78‐ and b96·11‐defined epitope distribution and GAD65 enzyme activity in sera from four cohorts with very high GADA titres: T1D patients (n = 7), GAD‐alum‐treated T1D patients (n = 9), T1D high‐risk individuals (n = 6) and SPS patients (n = 12). SPS patients showed significantly higher GADA levels and inhibited the in‐vitro GAD65 enzyme activity more strongly compared to the other groups. A higher binding frequency to the b78‐defined epitope was found in the SPS group compared to T1D and GAD‐alum individuals, whereas no differences were detected for the b96·11‐defined epitope. GADA IgG1–4 subclass levels did not differ between the groups, but SPS patients had higher IgG2 and lower IgG4 distribution more frequently. In conclusion, the in‐vitro GADA phenotypes from SPS patients differed from the T1D‐ and high‐risk groups, and GAD‐alum treatment did not induce SPS‐associated phenotypes. However, occasional overlap between the groups exists, and caution is indicated when drawing conclusions to health or disease status.

GAD autoantibody epitope pattern after GAD-alum treatment in children and adolescents with type 1 diabetes.

Skoglund C, Chéramy M, Casas R, Ludvigsson J, Hampe CS. GAD autoantibody epitope pattern after GAD‐alum treatment in children and adolescents with type 1 diabetes.

GAD-specific T cells are induced by GAD-alum treatment in Type-1 diabetes patients

Administration of Glutamic Acid Decarboxylase (GAD)65 formulated in aluminium hydroxide preserved insulin secretion in a phase II trial in recent onset Type 1 Diabetes. A subsequent European phase III trial was closed at 15months after failing to reach primary endpoint, but the majority of the Swedish patients completed the 21months follow-up. We studied the frequencies and phenotype of T cells, suppressive capacity of Tregs, GAD65-induced proliferation, and frequencies of T cells with a GAD65-specific TCR in Swedes participating in the trial. Stimulation with GAD65 induced activated T cells and also cells with a suppressive phenotype. Activated GAD65-specific effector T cells were detected by tetramer staining while the frequency of GAD65-specific Treg was not affected by the treatment. Additional doses of GAD-alum increased frequencies of CD25+CD127+, but had no effect on CD25hiCD127lo. Our findings indicate that GAD-alum treatment primarily induced activated T cells. GAD65-specific cells were mainly of activated phenotype.