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CD4(+) T cells from glutamic acid decarboxylase (GAD)65-specific T cell receptor transgenic mice are not diabetogenic and can delay diabetes transfer.

Tarbell KV, Lee M, Ranheim E, Chao CC, Sanna M, Kim SK, Dickie P, Teyton L, Davis M, McDevitt H - J. Exp. Med. (2002)

Bottom Line: However, the exact role of GAD65-specific T cells in diabetes pathogenesis is unclear.Lymphocytes from these TCR transgenic mice proliferate and make interferon gamma, interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, and IL-10 when stimulated in vitro with GAD65 peptide 286-300, yet these TCR transgenic animals do not spontaneously develop diabetes, and insulitis is virtually undetectable.This data suggests that GAD65 peptide 286-300-specific T cells have disease protective capacity and are not pathogenic.

View Article: PubMed Central - PubMed

Affiliation: Program in Immunology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Glutamic acid decarboxylase (GAD)65 is an early and important antigen in both human diabetes mellitus and the nonobese diabetic (NOD) mouse. However, the exact role of GAD65-specific T cells in diabetes pathogenesis is unclear. T cell responses to GAD65 occur early in diabetes pathogenesis, yet only one GAD65-specific T cell clone of many identified can transfer diabetes. We have generated transgenic mice on the NOD background expressing a T cell receptor (TCR)-specific for peptide epitope 286-300 (p286) of GAD65. These mice have GAD65-specific CD4(+) T cells, as shown by staining with an I-A(g7)(p286) tetramer reagent. Lymphocytes from these TCR transgenic mice proliferate and make interferon gamma, interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, and IL-10 when stimulated in vitro with GAD65 peptide 286-300, yet these TCR transgenic animals do not spontaneously develop diabetes, and insulitis is virtually undetectable. Furthermore, in vitro activated CD4 T cells from GAD 286 TCR transgenic mice express higher levels of CTL-associated antigen (CTLA)-4 than nontransgenic littermates. CD4(+) T cells, or p286-tetramer(+)CD4(+) Tcells, from GAD65 286-300-specific TCR transgenic mice delay diabetes induced in NOD.scid mice by diabetic NOD spleen cells. This data suggests that GAD65 peptide 286-300-specific T cells have disease protective capacity and are not pathogenic.

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Activation and cytokine production by lymphocytes from G286 mice. Lymph node cells from G286 or NOD mice were activated in vitro with the indicated concentration of p286. Proliferation for total LN cells (A), or purified populations (B) was determined by 3[H]thymidine incorporation. In B, CD4+ p286 tetramer-positive and CD4+ p286 tetramer-negative cells were sorted and activated with 20 μg/ml p286 and irradiated NOD spleen cells; 3[H]thymidine was added for the last 16 of a 96 h incubation. Results are expressed as average stimulation index. The average background counts for the no antigen controls were 1,396 cpm (A) and 58 cpm (B). (C) Expression of IL-2 at 48 h (top), IFN-γ at 62 h (middle), and IL-10 at 48 h (bottom) was determined by ELISA as described in Methods. (D) T cells expressing p286-specific TCR are activated in vivo by p286 peptide. 5-wk-old female G286 mice or transgene negative littermates were immunized in the footpad with either CFA or CFA with 10 μg/ml p286. 60 h later, cells from the draining lymph node were harvested. CD4+ gated cells are shown with p286 tetramer-PE (y axis), and either CD44-FITC or CD62L-FITC (x axis).
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fig4: Activation and cytokine production by lymphocytes from G286 mice. Lymph node cells from G286 or NOD mice were activated in vitro with the indicated concentration of p286. Proliferation for total LN cells (A), or purified populations (B) was determined by 3[H]thymidine incorporation. In B, CD4+ p286 tetramer-positive and CD4+ p286 tetramer-negative cells were sorted and activated with 20 μg/ml p286 and irradiated NOD spleen cells; 3[H]thymidine was added for the last 16 of a 96 h incubation. Results are expressed as average stimulation index. The average background counts for the no antigen controls were 1,396 cpm (A) and 58 cpm (B). (C) Expression of IL-2 at 48 h (top), IFN-γ at 62 h (middle), and IL-10 at 48 h (bottom) was determined by ELISA as described in Methods. (D) T cells expressing p286-specific TCR are activated in vivo by p286 peptide. 5-wk-old female G286 mice or transgene negative littermates were immunized in the footpad with either CFA or CFA with 10 μg/ml p286. 60 h later, cells from the draining lymph node were harvested. CD4+ gated cells are shown with p286 tetramer-PE (y axis), and either CD44-FITC or CD62L-FITC (x axis).

Mentions: To show that T cells from G286 mice were functional and not tolerant to peptide stimulation, lymph node and spleen cells were isolated and stimulated with p286. The peak stimulation index (SI), as determined by 3[H]thymidine incorporation, was 17 for lymph node cells, and occurred at the 10 μg/ml peptide dose (Fig. 4 A). G286 T cells also responded to GAD65 protein, stimulation index = 7 (unpublished data). When CD4+ T cells from G286 mice were sorted into tetramer-positive and tetramer-negative populations, only the tetramer-positive cells showed significant proliferation (Fig. 4 B). No IL-4 or IL-5 was detected in response to p286 in either the nontransgenic or G286 mice at any dose (unpublished data). Lymph node cells from G286 mice produced TNF-α, IL-2, IFN-γ, and IL-10 in response to p286 (Fig. 4 C and unpublished data). The levels of IFN-γ produced were high; in one experiment, an antigen dose of 15 μg/ml elicited 265 ng/ml of IFN-γ at 48 h. Lymphocytes from nontransgenic littermates produced no detectable IFN-γ, TNF-α, IL-2, or IL-10 in response to p286 (unpublished data). In conclusion, T cells from G286 mice are functional and respond in vitro to their cognate antigen with proliferation and cytokine production.


CD4(+) T cells from glutamic acid decarboxylase (GAD)65-specific T cell receptor transgenic mice are not diabetogenic and can delay diabetes transfer.

Tarbell KV, Lee M, Ranheim E, Chao CC, Sanna M, Kim SK, Dickie P, Teyton L, Davis M, McDevitt H - J. Exp. Med. (2002)

Activation and cytokine production by lymphocytes from G286 mice. Lymph node cells from G286 or NOD mice were activated in vitro with the indicated concentration of p286. Proliferation for total LN cells (A), or purified populations (B) was determined by 3[H]thymidine incorporation. In B, CD4+ p286 tetramer-positive and CD4+ p286 tetramer-negative cells were sorted and activated with 20 μg/ml p286 and irradiated NOD spleen cells; 3[H]thymidine was added for the last 16 of a 96 h incubation. Results are expressed as average stimulation index. The average background counts for the no antigen controls were 1,396 cpm (A) and 58 cpm (B). (C) Expression of IL-2 at 48 h (top), IFN-γ at 62 h (middle), and IL-10 at 48 h (bottom) was determined by ELISA as described in Methods. (D) T cells expressing p286-specific TCR are activated in vivo by p286 peptide. 5-wk-old female G286 mice or transgene negative littermates were immunized in the footpad with either CFA or CFA with 10 μg/ml p286. 60 h later, cells from the draining lymph node were harvested. CD4+ gated cells are shown with p286 tetramer-PE (y axis), and either CD44-FITC or CD62L-FITC (x axis).
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fig4: Activation and cytokine production by lymphocytes from G286 mice. Lymph node cells from G286 or NOD mice were activated in vitro with the indicated concentration of p286. Proliferation for total LN cells (A), or purified populations (B) was determined by 3[H]thymidine incorporation. In B, CD4+ p286 tetramer-positive and CD4+ p286 tetramer-negative cells were sorted and activated with 20 μg/ml p286 and irradiated NOD spleen cells; 3[H]thymidine was added for the last 16 of a 96 h incubation. Results are expressed as average stimulation index. The average background counts for the no antigen controls were 1,396 cpm (A) and 58 cpm (B). (C) Expression of IL-2 at 48 h (top), IFN-γ at 62 h (middle), and IL-10 at 48 h (bottom) was determined by ELISA as described in Methods. (D) T cells expressing p286-specific TCR are activated in vivo by p286 peptide. 5-wk-old female G286 mice or transgene negative littermates were immunized in the footpad with either CFA or CFA with 10 μg/ml p286. 60 h later, cells from the draining lymph node were harvested. CD4+ gated cells are shown with p286 tetramer-PE (y axis), and either CD44-FITC or CD62L-FITC (x axis).
Mentions: To show that T cells from G286 mice were functional and not tolerant to peptide stimulation, lymph node and spleen cells were isolated and stimulated with p286. The peak stimulation index (SI), as determined by 3[H]thymidine incorporation, was 17 for lymph node cells, and occurred at the 10 μg/ml peptide dose (Fig. 4 A). G286 T cells also responded to GAD65 protein, stimulation index = 7 (unpublished data). When CD4+ T cells from G286 mice were sorted into tetramer-positive and tetramer-negative populations, only the tetramer-positive cells showed significant proliferation (Fig. 4 B). No IL-4 or IL-5 was detected in response to p286 in either the nontransgenic or G286 mice at any dose (unpublished data). Lymph node cells from G286 mice produced TNF-α, IL-2, IFN-γ, and IL-10 in response to p286 (Fig. 4 C and unpublished data). The levels of IFN-γ produced were high; in one experiment, an antigen dose of 15 μg/ml elicited 265 ng/ml of IFN-γ at 48 h. Lymphocytes from nontransgenic littermates produced no detectable IFN-γ, TNF-α, IL-2, or IL-10 in response to p286 (unpublished data). In conclusion, T cells from G286 mice are functional and respond in vitro to their cognate antigen with proliferation and cytokine production.

Bottom Line: However, the exact role of GAD65-specific T cells in diabetes pathogenesis is unclear.Lymphocytes from these TCR transgenic mice proliferate and make interferon gamma, interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, and IL-10 when stimulated in vitro with GAD65 peptide 286-300, yet these TCR transgenic animals do not spontaneously develop diabetes, and insulitis is virtually undetectable.This data suggests that GAD65 peptide 286-300-specific T cells have disease protective capacity and are not pathogenic.

View Article: PubMed Central - PubMed

Affiliation: Program in Immunology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Glutamic acid decarboxylase (GAD)65 is an early and important antigen in both human diabetes mellitus and the nonobese diabetic (NOD) mouse. However, the exact role of GAD65-specific T cells in diabetes pathogenesis is unclear. T cell responses to GAD65 occur early in diabetes pathogenesis, yet only one GAD65-specific T cell clone of many identified can transfer diabetes. We have generated transgenic mice on the NOD background expressing a T cell receptor (TCR)-specific for peptide epitope 286-300 (p286) of GAD65. These mice have GAD65-specific CD4(+) T cells, as shown by staining with an I-A(g7)(p286) tetramer reagent. Lymphocytes from these TCR transgenic mice proliferate and make interferon gamma, interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, and IL-10 when stimulated in vitro with GAD65 peptide 286-300, yet these TCR transgenic animals do not spontaneously develop diabetes, and insulitis is virtually undetectable. Furthermore, in vitro activated CD4 T cells from GAD 286 TCR transgenic mice express higher levels of CTL-associated antigen (CTLA)-4 than nontransgenic littermates. CD4(+) T cells, or p286-tetramer(+)CD4(+) Tcells, from GAD65 286-300-specific TCR transgenic mice delay diabetes induced in NOD.scid mice by diabetic NOD spleen cells. This data suggests that GAD65 peptide 286-300-specific T cells have disease protective capacity and are not pathogenic.

Show MeSH
Related in: MedlinePlus