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GAD65 An Immunomodulator in Type 1 Diabetes
Linköping University, Department of Clinical and Experimental Medicine, Pediatrics. Linköping University, Faculty of Health Sciences.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Type 1 diabetes (T1D) is caused by a deficiency of insulin as a result of an autoimmune destruction of the pancreatic ² -cells. A possibility to preserve remaining ² -cells in children with newly diagnosed T1D is of great importance since sustained ² -cell function is recognized to result in reduced end-organ complications. Glutamic acid decarboxylase 65 (GAD65) is one of the major antigens targeted by self-reactive T cells in T1D, and immunomodulation with GAD65 formulated in aluminum (GAD-alum) has been considered both in prevention and treatment of T1D. Results from a Phase II trial have shown clinical effect of subcutaneous injections with GAD-alum, this was unfortunately not fully confirmed in the following larger Phase III trial which therefore was closed after 15 months. The general aim of this thesis was to study the immunomodulatory effect of GAD-alum-treatment in children with T1D participating in the Phase II and Phase III trials. We hypothesized that treatment with GAD-alum contributes to the preservation of residual insulin secretion through deviation of the GAD65-specific immune response from a destructive to a protective process, accompanied by a shift from T helper (Th) 1 towards a predominant Th2 profile. In the Phase II trial, GAD-alum-treated patients responded with an early GAD65-specific Th2 skewed cytokine secretion, with highest IL-5 and IL-13 secretion in clinical responders. Also, the CCR4/CCR5 ratio indicating balance between Th2/Tc2 and Th1/Tc1 responses, increased in treated patients. The recall response to GAD65 was characterized by a wide range of cytokines, but the relative contribution of each cytokine suggests a shift towards a more pronounced Th2-associated profile over time. Induction of a CD4+ cell subset upon GAD65-stimulation 4 years after treatment, suggesting clonal expansion of the memory T-cell compartment upon antigen re-challenge, was seen in parallel to a persistent GAD65-specific cytokine response. Finally, even if the phase III trial failed to reach the primary endpoint at 15 months, a subgroup analysis showed that the treatment had an effect on preservation of residual insulin secretion, but the effect was not seen until after 30 months. Taken together, these results suggest that GAD-alum treatment might exert its effect through induction of an early Th2 skewed immune response which tends to deviate away from a destructive Th1/Tc1 response upon GAD65 re-challenge, and generation of GAD65-specific memory T cells that produce cytokines and exert effector responses which may be important for regulating GAD65 immunity. Continued research to better understand how immunomodulation with autoantigen modifies T-cell responses and also which patients are suitable for treatment, is crucial for optimizing future intervention trials using ² -cell antigens.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. , 81 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1310
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-77747ISBN: 978-91-7519-888-0 (print)OAI: oai:DiVA.org:liu-77747DiVA: diva2:528715
Public defence
2012-06-01, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2012-05-28 Created: 2012-05-28 Last updated: 2012-05-28Bibliographically approved
List of papers
1. Early induction of GAD(65)-reactive Th2 response in type 1 diabetic children treated with alum-formulated GAD(65)
Open this publication in new window or tab >>Early induction of GAD(65)-reactive Th2 response in type 1 diabetic children treated with alum-formulated GAD(65)
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2010 (English)In: Diabetes/Metabolism Research Reviews, ISSN 1520-7552, E-ISSN 1520-7560, Vol. 26, no 7, 559-568 p.Article in journal (Refereed) Published
Abstract [en]

Background We have previously shown that two injections of 20 mu g alum-formulated glutamic acid decarboxylase 65 (GAD(65)) (GAD-alum; Diamyd (R)) in children with recent-onset type 1 diabetes lead to preservation of residual insulin secretion. In vitro cytokine production at the 15 months follow-up indicated immunomodulation. In the present study, we took advantage of peripheral blood mononuclear cells, cryopreserved during early follow-ups, to investigate whether the immunomodulatory effect of GAD-alum was apparent earlier after treatment, preceding the changes previously reported at 15 months.<p>Methods Peripheral blood mononuclear cells from 70 type 1 diabetic children, randomly assigned GAD-alum (n = 35) or placebo (n = 35), that had been frozen at baseline (n = 27) and after 1 (n = 58), 3 (n = 67) and 9 (n = 66) months, were stimulated in vitro with GAD(65), tyrosine phosphatase-like protein IA-2 peptide, insulin peptide, GAD-alum, alum formulation or phytohaemagglutinin. Interleukin (IL)-5, -6, -10, -12, -13, -17, tumour necrosis factor and interferon-gamma were measured in cell supernatants and serum samples using Luminex. Expression of FOXP3 and transforming growth factor-beta was determined by real-time reverse transcription polymerase chain reaction.</p><p>Results Already 1 month after the first injection, GAD(65)-induced IL-5 and IL-13 together with FOXP3 were enhanced in GAD-alum-treated patients compared to those with placebo. The in vitro response at 3 and 9 months was characterized by a broader range of cytokines in the treated group. Notably, only the T-helper 2-associated cytokines IL-5 and IL-13 together with FOXP3 increased continuously over time.</p><p>Conclusions Treatment with GAD-alum in type 1 diabetic children induced an early T-helper 2 immune enhanced response to GAD(65), followed by a wider spectrum of cytokines at 3 and 9 months. Copyright (C) 2010 John Wiley &amp; Sons, Ltd.</p>

Place, publisher, year, edition, pages
John Wiley and Sons, 2010
Keyword
GAD65, Immunotherapy, Th1/Th2 Immune Response, Immunomodulation, Cytokines
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-52141 (URN)10.1002/dmrr.1126 (DOI)000283399000007 ()
Available from: 2009-12-07 Created: 2009-12-07 Last updated: 2017-12-12
2. Decreased GAD(65) -specific Th1/Tc1 phenotype in children with Type 1 diabetes treated with GAD-alum.
Open this publication in new window or tab >>Decreased GAD(65) -specific Th1/Tc1 phenotype in children with Type 1 diabetes treated with GAD-alum.
2012 (English)In: Diabetic Medicine, ISSN 0742-3071, E-ISSN 1464-5491, Vol. 29, no 10, 1272-1278 p.Article in journal (Refereed) Published
Abstract [en]

Aim  The balance between T helper cell subsets is an important regulator of the immune system and is often examined after immune therapies. We aimed to study the immunomodulatory effect of glutamic acid decarboxylase (GAD) 65 formulated with aluminium hydroxide (GAD-alum) in children with Type 1 diabetes, focusing on chemokines and their receptors. Methods  Blood samples were collected from 70 children with Type 1 diabetes included in a phase II clinical trial with GAD-alum. Expression of CC chemokine receptor 5 (CCR5) and CCR4 was analysed on CD4+ and CD8+ lymphocytes after in vitro stimulation with GAD(65) using flow cytometry, and secretion of the chemokines CCL2, CCL3 and CCL4 was detected in peripheral blood mononuclear cell supernatants with Luminex. Results  Expression of Th1-associated CCR5 was down-regulated following antigen challenge, together with an increased CCR4/CCR5 ratio and CCL2 secretion in GAD-alum-treated patients, but not in the placebo group. Conclusion  Our results suggest that GAD-alum treatment has induced a favourable immune modulation associated with decreased Th1/Tc1 phenotypes upon antigen re-challenge, which may be of importance for regulating GAD(65) immunity. © 2012 The Authors. Diabetic Medicine © 2012 Diabetes UK.

Place, publisher, year, edition, pages
Wiley-Blackwell, 2012
Keyword
Type 1 diabetes, GAD(65), immunomodulation, chemokines, chemokine receptors
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-77745 (URN)10.1111/j.1464-5491.2012.03710.x (DOI)000308960400014 ()22587593 (PubMedID)
Note

funding agencies|Swedish Research Council|K2008-55x-20652-01-3|Swedish Child Diabetes Foundation (Barndiabetesfonden) Diamyd Medical||Medical Research Council of Southeast Sweden||

Available from: 2012-05-28 Created: 2012-05-28 Last updated: 2017-12-07
3. Long-Lasting Immune Responses 4 Years after GAD-Alum Treatment in Children with Type 1 Diabetes
Open this publication in new window or tab >>Long-Lasting Immune Responses 4 Years after GAD-Alum Treatment in Children with Type 1 Diabetes
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 12Article in journal (Refereed) Published
Abstract [en]

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 GAD(65). Frequencies of naive, central and effector memory CD4+ and CD8+ T cells were measured, together with cytokine secretion, proliferation, gene expression and serum GAD(65) 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 GAD(65), but not with control antigens, compared with placebo subjects. GAD(65)-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 GAD(65) enzymatic activity. In conclusion, memory T- and B-cell responses persist 4 years after GAD-alum-treatment. In parallel to a GAD(65)-induced T-cell activation, our results show induction of T-cell inhibitory pathways important for regulating the GAD(65) immunity.

Place, publisher, year, edition, pages
Public Library of Science, 2011
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-74156 (URN)10.1371/journal.pone.0029008 (DOI)000298366600057 ()
Note
Funding Agencies|Swedish Research Council|K2008-55x-20652-01-3|Swedish Child Diabetes Foundation (Barndiabetesfonden)||Medical Research Council of Southeast Sweden||Juvenile Diabetes Research Foundation (JDRF)|1-2008-106|Ile-de-France CODDIM||Inserm Avenir Program||Available from: 2012-01-20 Created: 2012-01-20 Last updated: 2017-12-08
4. Preserved C-peptide 30 months after GAD-alum treatment of children and adolescents with recent-onset type 1 diabetes, and its relation to immune markers
Open this publication in new window or tab >>Preserved C-peptide 30 months after GAD-alum treatment of children and adolescents with recent-onset type 1 diabetes, and its relation to immune markers
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Glutamic acid decarboxylase 65 kDa isoform (GAD65) is a major autoantigen in type 1 diabetes (T1D). Although alum-formulated GAD65 (GAD-alum) induced preservation of residual insulin secretion in a previous clinical Phase II trial, recent Phase II and Phase III trials failed to reach their primary end-points. The European Phase III trial was therefore closed after 15 months, and the 30 months follow-up period was completed only for a minority of the patients. This study aimed to assess whether GAD-alum preserved β-cell function in those recent-onset T1D patients who completed their 30 months visit in the European Phase III trial, and to characterize their GAD65-induced cytokine secretion and proliferation. Peripheral blood mononuclear cells (PBMC) were isolated at baseline and after 1, 3, 9, 15 and 21 months from the 148 Swedish subjects included in the Phase III GAD-alum trial, and also at 30 months from 45 patients who had reached the final visit before the trial was closed. Patients had been randomly assigned into three arms: 4 doses of GAD-alum (4D), 2 doses of GAD-alum followed by two doses of placebo (2D), or 4 doses of placebo. Cytokine secretion was detected in cell culture supernatants by Luminex, after 7 days of in vitro culture. Cell proliferation was determined by 3H thymidine incorporation assay. Fasting and stimulated C-peptide was analysed in serum.

Patients treated with 2 doses of GAD-alum had less decline of both fasting (p=0.040) and stimulated C-peptide (p=0.012) after 30 months, and a larger proportion of these patients preserved >25% of their initial stimulated C-peptide AUC compared to placebo (p=0.012). Both 2D and 4D patients showed increased PBMC proliferation to GAD65 and a cytokine profile that tended to switch towards a more predominant Th2 associated profile over time.

The results support the concept of GAD-alum treatment, but no specific immune markers have been identified.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-77746 (URN)
Available from: 2012-05-28 Created: 2012-05-28 Last updated: 2012-05-28Bibliographically approved

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