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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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Characterization of G286 mice. (A) Integration of TCR-Vα and -Vβ constructs is shown by genomic PCR. (Left) DNA amplified with TCR-α specific primers from transgene positive (lane 1) and transgene negative (lane 2) mice. (Right) DNA amplified with TCR-β specific primers from transgene positive (lanes 3 and 4) and transgene negative (lane 5) mice. (B) Staining of peripheral blood lymphocytes with antibodies against Vβ8 (x axis) and CD4 (y axis) for NOD littermate (left) and transgene positive (right). (C) Staining of spleen cells from NOD (top) or G286 mice (bottom) with I-Ag7-286 tetramer and CD4 or CD8 (right). As a control, cells were also stained with an I-Ag7 tetramer containing peptide 323–339 of OVA (left). (D) Thymocytes from NOD (left) or G286 (right) mice were stained with antibodies against CD4 and CD8. The percentage of cells in different boxed gates are shown.
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fig1: Characterization of G286 mice. (A) Integration of TCR-Vα and -Vβ constructs is shown by genomic PCR. (Left) DNA amplified with TCR-α specific primers from transgene positive (lane 1) and transgene negative (lane 2) mice. (Right) DNA amplified with TCR-β specific primers from transgene positive (lanes 3 and 4) and transgene negative (lane 5) mice. (B) Staining of peripheral blood lymphocytes with antibodies against Vβ8 (x axis) and CD4 (y axis) for NOD littermate (left) and transgene positive (right). (C) Staining of spleen cells from NOD (top) or G286 mice (bottom) with I-Ag7-286 tetramer and CD4 or CD8 (right). As a control, cells were also stained with an I-Ag7 tetramer containing peptide 323–339 of OVA (left). (D) Thymocytes from NOD (left) or G286 (right) mice were stained with antibodies against CD4 and CD8. The percentage of cells in different boxed gates are shown.

Mentions: Incorporation and expression of the transgene was assessed by genomic PCR and flow cytometry, respectively. Transgenic TCR-α and -β specific primers amplify PCR product from genomic DNA isolated from transgene-positive but not transgene-negative littermates (Fig. 1 A). Because a Vβ1-specific antibody is not available, expression of the TCR-β chain transgene was determined by a lack of expression of other Vβ proteins due to allelic exclusion. In NOD mice, the normal expression of CD4+ Vβ8+ cells is 8–12% of peripheral blood lymphocytes, but in G286 mice, the percentage of CD4+Vβ8+ cells is <0.5% of PBLs (Fig. 1 B). Vβ5 staining gave similar results (unpublished data). This suggests that 90–95% of the T cells express the transgenic β chain. The α chain of this TCR is Vα4.5, for which there also is not a specific antibody. Because allelic exclusion is not as complete for TCR-α chain, one cannot assess expression of the TCR-α transcript using this same approach.


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)

Characterization of G286 mice. (A) Integration of TCR-Vα and -Vβ constructs is shown by genomic PCR. (Left) DNA amplified with TCR-α specific primers from transgene positive (lane 1) and transgene negative (lane 2) mice. (Right) DNA amplified with TCR-β specific primers from transgene positive (lanes 3 and 4) and transgene negative (lane 5) mice. (B) Staining of peripheral blood lymphocytes with antibodies against Vβ8 (x axis) and CD4 (y axis) for NOD littermate (left) and transgene positive (right). (C) Staining of spleen cells from NOD (top) or G286 mice (bottom) with I-Ag7-286 tetramer and CD4 or CD8 (right). As a control, cells were also stained with an I-Ag7 tetramer containing peptide 323–339 of OVA (left). (D) Thymocytes from NOD (left) or G286 (right) mice were stained with antibodies against CD4 and CD8. The percentage of cells in different boxed gates are shown.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196059&req=5

fig1: Characterization of G286 mice. (A) Integration of TCR-Vα and -Vβ constructs is shown by genomic PCR. (Left) DNA amplified with TCR-α specific primers from transgene positive (lane 1) and transgene negative (lane 2) mice. (Right) DNA amplified with TCR-β specific primers from transgene positive (lanes 3 and 4) and transgene negative (lane 5) mice. (B) Staining of peripheral blood lymphocytes with antibodies against Vβ8 (x axis) and CD4 (y axis) for NOD littermate (left) and transgene positive (right). (C) Staining of spleen cells from NOD (top) or G286 mice (bottom) with I-Ag7-286 tetramer and CD4 or CD8 (right). As a control, cells were also stained with an I-Ag7 tetramer containing peptide 323–339 of OVA (left). (D) Thymocytes from NOD (left) or G286 (right) mice were stained with antibodies against CD4 and CD8. The percentage of cells in different boxed gates are shown.
Mentions: Incorporation and expression of the transgene was assessed by genomic PCR and flow cytometry, respectively. Transgenic TCR-α and -β specific primers amplify PCR product from genomic DNA isolated from transgene-positive but not transgene-negative littermates (Fig. 1 A). Because a Vβ1-specific antibody is not available, expression of the TCR-β chain transgene was determined by a lack of expression of other Vβ proteins due to allelic exclusion. In NOD mice, the normal expression of CD4+ Vβ8+ cells is 8–12% of peripheral blood lymphocytes, but in G286 mice, the percentage of CD4+Vβ8+ cells is <0.5% of PBLs (Fig. 1 B). Vβ5 staining gave similar results (unpublished data). This suggests that 90–95% of the T cells express the transgenic β chain. The α chain of this TCR is Vα4.5, for which there also is not a specific antibody. Because allelic exclusion is not as complete for TCR-α chain, one cannot assess expression of the TCR-α transcript using this same approach.

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