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Normal incidence of diabetes in NOD mice tolerant to glutamic acid decarboxylase.

Jaeckel E, Klein L, Martin-Orozco N, von Boehmer H - J. Exp. Med. (2003)

Bottom Line: Several attempts of inducing GAD-specific recessive tolerance to support this hypothesis have failed.Here we report on successful tolerance induction by expressing a modified form of GAD under control of the invariant chain promoter resulting in efficient epitope display.In spite of specific tolerance insulitis and diabetes occurred with normal kinetics indicating that GAD is not an essential autoantigen in the pathogenesis of diabetes.

View Article: PubMed Central - PubMed

Affiliation: Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Experiments in nonobese diabetic (NOD) mice that lacked expression of glutamic acid decarboxylase (GAD) in beta cells have suggested that GAD represents an autoantigen essential for initiating and maintaining the diabetogenic immune response. Several attempts of inducing GAD-specific recessive tolerance to support this hypothesis have failed. Here we report on successful tolerance induction by expressing a modified form of GAD under control of the invariant chain promoter resulting in efficient epitope display. In spite of specific tolerance insulitis and diabetes occurred with normal kinetics indicating that GAD is not an essential autoantigen in the pathogenesis of diabetes.

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T cell responses of GAD65 transgenic animals against GAD65 and its peptide epitopes. (A) Proliferation assay of draining lymph node cells of 10-wk-old NOD mice 8 d after immunization with GAD65 in complete (black bars) and incomplete Freund's adjuvants (open bars) into foot pads. Representative result from five independent experiments. (B and C) Proliferative response of draining lymph node cells of NOD control mice (black bars) and two GAD65 transgenic lines (gray bars: line 14; open bars: line 29) to GAD65 and its peptide epitopes (all 10 μg/ml) 8 d after immunization with GAD65 in CFA. CF, culture filtrate protein of M. tuberculosis as positive control. (C) Titration of the positive proliferative recall responses as seen in B. (▪ NOD controls; ⋄ GAD65tg. line 14; Δ GAD65tg. line 29).
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fig2: T cell responses of GAD65 transgenic animals against GAD65 and its peptide epitopes. (A) Proliferation assay of draining lymph node cells of 10-wk-old NOD mice 8 d after immunization with GAD65 in complete (black bars) and incomplete Freund's adjuvants (open bars) into foot pads. Representative result from five independent experiments. (B and C) Proliferative response of draining lymph node cells of NOD control mice (black bars) and two GAD65 transgenic lines (gray bars: line 14; open bars: line 29) to GAD65 and its peptide epitopes (all 10 μg/ml) 8 d after immunization with GAD65 in CFA. CF, culture filtrate protein of M. tuberculosis as positive control. (C) Titration of the positive proliferative recall responses as seen in B. (▪ NOD controls; ⋄ GAD65tg. line 14; Δ GAD65tg. line 29).

Mentions: GAD65-specific tolerance was tested in nonprimed animals (proliferation-assay/ELISPOT/tetramer-staining) and after immunization with recombinant murine GAD65 produced in an E. coli system. Unprimed cells from spleen and pancreatic lymph nodes displayed no consistent response upon culture in the presence of GAD65 or its peptide epitopes as measured by proliferation or ELISPOT analysis. Similar difficulties in obtaining reproducible “spontaneous” responses of nonprimed T cells have been reported by other groups (25, 26). To achieve a more reliable assessment of GAD-specific T cell responses animals were immunized with recombinant GAD65 protein produced in an E. coli system. Proliferative recall responses were then tested using either GAD65 peptides or whole protein. GAD65 protein used for restimulation was produced in a baculoviral expression system to prevent recall responses to contaminants from the E. coli preparation used as an immunogen. In nontransgenic mice, the qualitative spectrum (responses against GAD65 protein and p206, p217, p221, p286) of the immune response was similar after immunization with GAD65 in either complete or incomplete Freund's adjuvants ruling out an alteration of the GAD65 epitope hierarchy by the use of agents stimulating APCs (Fig. 2 A). GAD65tg mice exhibited tolerance to the entire GAD65 protein as well as to all tested peptide epitopes (Fig. 2, B and C). Although GAD65-specific tolerance in NOD mice could only be induced by transgenic expression of a modified form of GAD65, tolerance was seen in recall assays using the entire, unmodified GAD65 protein, thereby excluding that the modifications altered epitopes that can be derived from the wild-type protein. No responses to epitopes p78, p246, p340, p479, p509, and p530 were detected in either immunized GAD transgenic or NOD mice indicating that these epitopes are only recognized after specific immunization/stimulation with the respective peptides but are not part of the normal T cell response to the GAD protein.


Normal incidence of diabetes in NOD mice tolerant to glutamic acid decarboxylase.

Jaeckel E, Klein L, Martin-Orozco N, von Boehmer H - J. Exp. Med. (2003)

T cell responses of GAD65 transgenic animals against GAD65 and its peptide epitopes. (A) Proliferation assay of draining lymph node cells of 10-wk-old NOD mice 8 d after immunization with GAD65 in complete (black bars) and incomplete Freund's adjuvants (open bars) into foot pads. Representative result from five independent experiments. (B and C) Proliferative response of draining lymph node cells of NOD control mice (black bars) and two GAD65 transgenic lines (gray bars: line 14; open bars: line 29) to GAD65 and its peptide epitopes (all 10 μg/ml) 8 d after immunization with GAD65 in CFA. CF, culture filtrate protein of M. tuberculosis as positive control. (C) Titration of the positive proliferative recall responses as seen in B. (▪ NOD controls; ⋄ GAD65tg. line 14; Δ GAD65tg. line 29).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2193961&req=5

fig2: T cell responses of GAD65 transgenic animals against GAD65 and its peptide epitopes. (A) Proliferation assay of draining lymph node cells of 10-wk-old NOD mice 8 d after immunization with GAD65 in complete (black bars) and incomplete Freund's adjuvants (open bars) into foot pads. Representative result from five independent experiments. (B and C) Proliferative response of draining lymph node cells of NOD control mice (black bars) and two GAD65 transgenic lines (gray bars: line 14; open bars: line 29) to GAD65 and its peptide epitopes (all 10 μg/ml) 8 d after immunization with GAD65 in CFA. CF, culture filtrate protein of M. tuberculosis as positive control. (C) Titration of the positive proliferative recall responses as seen in B. (▪ NOD controls; ⋄ GAD65tg. line 14; Δ GAD65tg. line 29).
Mentions: GAD65-specific tolerance was tested in nonprimed animals (proliferation-assay/ELISPOT/tetramer-staining) and after immunization with recombinant murine GAD65 produced in an E. coli system. Unprimed cells from spleen and pancreatic lymph nodes displayed no consistent response upon culture in the presence of GAD65 or its peptide epitopes as measured by proliferation or ELISPOT analysis. Similar difficulties in obtaining reproducible “spontaneous” responses of nonprimed T cells have been reported by other groups (25, 26). To achieve a more reliable assessment of GAD-specific T cell responses animals were immunized with recombinant GAD65 protein produced in an E. coli system. Proliferative recall responses were then tested using either GAD65 peptides or whole protein. GAD65 protein used for restimulation was produced in a baculoviral expression system to prevent recall responses to contaminants from the E. coli preparation used as an immunogen. In nontransgenic mice, the qualitative spectrum (responses against GAD65 protein and p206, p217, p221, p286) of the immune response was similar after immunization with GAD65 in either complete or incomplete Freund's adjuvants ruling out an alteration of the GAD65 epitope hierarchy by the use of agents stimulating APCs (Fig. 2 A). GAD65tg mice exhibited tolerance to the entire GAD65 protein as well as to all tested peptide epitopes (Fig. 2, B and C). Although GAD65-specific tolerance in NOD mice could only be induced by transgenic expression of a modified form of GAD65, tolerance was seen in recall assays using the entire, unmodified GAD65 protein, thereby excluding that the modifications altered epitopes that can be derived from the wild-type protein. No responses to epitopes p78, p246, p340, p479, p509, and p530 were detected in either immunized GAD transgenic or NOD mice indicating that these epitopes are only recognized after specific immunization/stimulation with the respective peptides but are not part of the normal T cell response to the GAD protein.

Bottom Line: Several attempts of inducing GAD-specific recessive tolerance to support this hypothesis have failed.Here we report on successful tolerance induction by expressing a modified form of GAD under control of the invariant chain promoter resulting in efficient epitope display.In spite of specific tolerance insulitis and diabetes occurred with normal kinetics indicating that GAD is not an essential autoantigen in the pathogenesis of diabetes.

View Article: PubMed Central - PubMed

Affiliation: Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

ABSTRACT
Experiments in nonobese diabetic (NOD) mice that lacked expression of glutamic acid decarboxylase (GAD) in beta cells have suggested that GAD represents an autoantigen essential for initiating and maintaining the diabetogenic immune response. Several attempts of inducing GAD-specific recessive tolerance to support this hypothesis have failed. Here we report on successful tolerance induction by expressing a modified form of GAD under control of the invariant chain promoter resulting in efficient epitope display. In spite of specific tolerance insulitis and diabetes occurred with normal kinetics indicating that GAD is not an essential autoantigen in the pathogenesis of diabetes.

Show MeSH
Related in: MedlinePlus