Immunization of non-obese diabetic (NOD) mice with glutamic acid decarboxylase-derived peptide 524–543 reduces cyclophosphamide-accelerated diabetes

Abstract

SUMMARYNOD mice constitute a model for studying the prevention of human autoimmune type 1 diabetes. Glutamic acid decarboxylase (GAD) could be a key antigen involved in this disease, and GAD65 peptide 524-543 has been implicated in early T cell response in young NOD mice. We performed two i.p. injections of GAD peptide 524-543 (100 ¹ g at each injection), together with Freund's incomplete adjuvant (FIA), into female NOD mice at 30 and 45 days old. Diabetes was accelerated 2 weeks later by a single injection of cyclophosphamide (CY), which acts against suppressive mechanisms. Treatment with GAD 524-543 peptide delayed the onset of diabetes and reduced its incidence (28% versus 60%; P < 0 . 001) compared with control mice injected with FIA alone, or GAD peptide 534-553, or an irrelevant peptide. In the same group, the severity of lymphocytic inflammation of pancreatic islets was reduced (P < 0 . 03). Up to 3 months after peptide injections, a strong splenocytic proliferative response occurred in immunized NOD mice against the immunizing peptide alone (but not against a panel of seven other GAD65-derived peptides). After peptide challenge of splenocytes in vitro, protection against CY-accelerated diabetes was associated with higher peptide-specific production of T helper type 2 (Th2)-associated interleukins 4 and 10, whereas Th1-associated interferon-gamma and IL-2 were proportionally less represented. During cotransfer, T splenocytes from GAD 524-543-immunized mice were able to reduce the capacity of T cells from diabetic donors to transfer the disease adoptively (P < 0 . 01), demonstrating the generation of cellular mechanisms that actively suppress the disease. It is concluded that immunization of NOD mice with GAD65 peptide 524-543 can counteract CYaccelerated diabetes, possibly through active cellular suppression linked to a shift of Th1/Th2 balance toward the production of Th2 cytokines such as IL-4 and IL-10. This study provides additional support for the notion that GAD, and more precisely its epitope 524-543, could be one of the key targets for the pathogenesis of type 1 diabetes in NOD mice, as well as for the efficacy of disease-specific peptide therapy in type 1 diabetes.

Regulatory and Effector CD4 T Cells in Nonobese Diabetic Mice Recognize Overlapping Determinants on Glutamic Acid Decarboxylase and Use DistinctVβGenes

“…Only 25% of the p524-treated mice became diabetic, compared with 88 -100% IDDM in the controls treated with IFA or peptide 167-181, respectively. Seven of 12 mice became diabetic after treatment with peptide p530 (Table III) and the 50% reduction in disease incidence in the GAD65(524 -543)-treated group (Table III) was similar to that seen in a previous report (14). Despite their strong immunogenicity in NOD mice, GAD65 peptides 206 -220 (5) and 167-181 (our unpublished data) afforded little to no amelioration from cyclophosphamide-induced IDDM (Table III), thus demonstrating that immunogenicity was not sufficient to provide protection from the accelerated form of IDDM.…”

“…Pretreatment with GAD65(524 -543) has been shown to protect NOD mice from spontaneous (1) and cyclophosphamide-induced IDDM (14), presumably by rendering the effector T cells tolerant. In this study, we show that while IDDM was reduced in NOD mice neonatally treated with GAD65 peptides p524, p530, or 524 -543, compared with PBS/IFA-treated animals, p524 was the most efficacious and significantly better at providing protection from disease (Table III).…”

Th1-like dominance in high-risk first-degree relatives of Type I diabetic patients

“…Active cellular suppression linked to a shift of the Th1:Th2 balance towards production of Th2 cytokines by immunisation of NOD mice with GAD 65 (a. a. 524±543) has been reported [17]. Epitope-specific therapy by T-cell epitopes of GAD 65 , especially amino acids 524±543 [16], is furthermore suggested to be of importance for the design of therapeutic approaches in human diabetes [41].…”

“…The sequence (a. a. 524±543) next to this epitope has been shown to be able to delay the onset of diabetes and reduce its incidence in non-obese diabetic (NOD) mice [17].…”

Processing and presentation of (pro)‐insulin in the MHC class II pathway: the generation of antigen‐based immunomodulators in the context of type 1 diabetes mellitus

“…The GAD-derived APL prevent the onset of EAD in NOD mice [128], and an islet antigen-derived APL downregulate the proliferation of a T cell clone [129]. The minimal T cell epitope (p57-67) from Imogen 38, an antigen from the mitochondria of islet cells (38 kDa), has been used to generate several APL.…”