Limits...
Deletion of the G6pc2 gene encoding the islet-specific glucose-6-phosphatase catalytic subunit-related protein does not affect the progression or incidence of type 1 diabetes in NOD/ShiLtJ mice.

Oeser JK, Parekh VV, Wang Y, Jegadeesh NK, Sarkar SA, Wong R, Lee CE, Pound LD, Hutton JC, Van Kaer L, O'Brien RM - Diabetes (2011)

Bottom Line: Insulitis was prominent in both groups, but whereas NOD/ShiLtJ G6pc2(+/+) islets contained CD8(+) T cells reactive to the G6pc2 NRP peptide, G6pc2 NRP-reactive T cells were absent in NOD/ShiLtJ G6pc2(-/-) islets.These results demonstrate that G6pc2 is an important driver for the selection and expansion of islet-reactive CD8(+) T cells infiltrating NOD/ShiLtJ islets.However, autoreactivity to G6pc2 is not essential for the emergence of autoimmune diabetes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

ABSTRACT

Objective: Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), now known as G6PC2, is a major target of autoreactive T cells implicated in the pathogenesis of type 1 diabetes in both mice and humans. This study aimed to determine whether suppression of G6p2 gene expression might therefore prevent or delay disease progression.

Research design and methods: G6pc2(-/-) mice were generated on the NOD/ShiLtJ genetic background, and glycemia was monitored weekly up to 35 weeks of age to determine the onset and incidence of diabetes. The antigen specificity of CD8(+) T cells infiltrating islets from NOD/ShiLtJ G6pc2(+/+) and G6pc2(-/-) mice at 12 weeks was determined in parallel.

Results: The absence of G6pc2 did not affect the time of onset, incidence, or sex bias of type 1 diabetes in NOD/ShiLtJ mice. Insulitis was prominent in both groups, but whereas NOD/ShiLtJ G6pc2(+/+) islets contained CD8(+) T cells reactive to the G6pc2 NRP peptide, G6pc2 NRP-reactive T cells were absent in NOD/ShiLtJ G6pc2(-/-) islets.

Conclusions: These results demonstrate that G6pc2 is an important driver for the selection and expansion of islet-reactive CD8(+) T cells infiltrating NOD/ShiLtJ islets. However, autoreactivity to G6pc2 is not essential for the emergence of autoimmune diabetes. The results remain consistent with previous studies indicating that insulin may be the primary autoimmune target, at least in NOD/ShiLtJ mice.

Show MeSH

Related in: MedlinePlus

Islet autoantigen–specific CD8+ T-cell responses in wild-type and G6pc2-deficient NOD/ShiLtJ mice. A and B: Islets were isolated from individual female wild-type or G6pc2−/− NOD/ShiLtJ mice at 12 weeks of age and cultured in the presence of interleukin-2 for 9 days. Cells were then stained with FITC-labeled anti-CD8α and PE-labeled MHC–class I tetramers loaded with the indicated peptides and analyzed by flow cytometry. Tetramers loaded with Flu-NP366–374 or Flu-NP147–155 were used as a negative control for tetramer staining. Data were electronically gated for CD8α-expressing cells. A: Representative flow cytometry data for one wild-type mouse and one G6pc2-deficient mouse. Numbers indicate the percentage of cells within the gated area. B: Summary of the flow cytometry data for four wild-type and four G6pc2-deficient mice from two experiments. C: Splenic and lymph-node cells were prepared from female wild-type or G6pc2−/− NOD/ShiLtJ mice at 12 weeks of age, and the frequency of NRP-V7–specific CD8+ T cells was determined by tetramer staining. Data were electronically gated for CD8α-expressing cells. Representative flow cytometry data are shown. D: Wild-type or G6pc2−/− NOD/ShiLtJ female mice were immunized with NRP-V7 peptide (100 μg per mouse) emulsified in complete Freund’s adjuvant. Two weeks later, splenic and draining lymph-node cells were prepared and stained with anti–CD8α-FITC and NRP-V7 or control Flu-NP tetramer and analyzed by flow cytometry. Data were electronically gated for CD8α-expressing cells. Representative flow cytometry data are shown. NS, not significant; WT, wild-type. (A high-quality color representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3198073&req=5

Figure 4: Islet autoantigen–specific CD8+ T-cell responses in wild-type and G6pc2-deficient NOD/ShiLtJ mice. A and B: Islets were isolated from individual female wild-type or G6pc2−/− NOD/ShiLtJ mice at 12 weeks of age and cultured in the presence of interleukin-2 for 9 days. Cells were then stained with FITC-labeled anti-CD8α and PE-labeled MHC–class I tetramers loaded with the indicated peptides and analyzed by flow cytometry. Tetramers loaded with Flu-NP366–374 or Flu-NP147–155 were used as a negative control for tetramer staining. Data were electronically gated for CD8α-expressing cells. A: Representative flow cytometry data for one wild-type mouse and one G6pc2-deficient mouse. Numbers indicate the percentage of cells within the gated area. B: Summary of the flow cytometry data for four wild-type and four G6pc2-deficient mice from two experiments. C: Splenic and lymph-node cells were prepared from female wild-type or G6pc2−/− NOD/ShiLtJ mice at 12 weeks of age, and the frequency of NRP-V7–specific CD8+ T cells was determined by tetramer staining. Data were electronically gated for CD8α-expressing cells. Representative flow cytometry data are shown. D: Wild-type or G6pc2−/− NOD/ShiLtJ female mice were immunized with NRP-V7 peptide (100 μg per mouse) emulsified in complete Freund’s adjuvant. Two weeks later, splenic and draining lymph-node cells were prepared and stained with anti–CD8α-FITC and NRP-V7 or control Flu-NP tetramer and analyzed by flow cytometry. Data were electronically gated for CD8α-expressing cells. Representative flow cytometry data are shown. NS, not significant; WT, wild-type. (A high-quality color representation of this figure is available in the online issue.)

Mentions: Diabetes in the NOD/ShiLtJ mouse results from the specific destruction of pancreatic β-cells by a cell-mediated process involving an autoreactive antigen-specific CD4+ and CD8+ T-cell attack in an environment where regulatory T-cell responses are perturbed (6). Molecularly defined targets of pathogenic CD8+ T cells have been well established (6) and include an H-2Kd–restricted peptide of G6pc2206–214 and its mimotope NRP-V7 (6) and a H-2Kd–restricted peptide of the insulin B chain (insulin B15–23 and its variant INS-L9), both of which have been implicated in disease pathogenesis and both of which are recognized by an appreciable proportion of CD8+ T cells residing in islet infiltrates in NOD/ShiLtJ mice (6). We compared the prevalence of islet autoantigen-specific CD8+ T cells in islets isolated from 12-week-old wild-type and NOD/ShiLtJ G6pc2−/− mice. Islets were cultured for 9 days ex vivo in the presence of interleukin-2 to expand CD8+ T cells residing in them. Cells were then stained with a panel of peptide-loaded MHC–class I tetramers bearing the abovementioned G6pc2 and insulin peptides, or influenza peptides as negative controls, and analyzed by flow cytometry. As previously reported, CD8+ T cells reactive to NRP-V7 were the dominant population of CD8+ T cells in wild-type mouse islets, accompanied by a variable, lesser percentage of insulin B15–23–and MimA2-specific T cells (Fig. 4A and B). In contrast, G6pc2−/− mouse islets completely lacked CD8+ T cells reactive to NRP-V7 (Fig. 4A and B) but retained a high proportion of CD8+ T cells specific for insulin B (Fig. 4A and B). The CD8+ T cells in the islets of G6pc2−/− mice were functionally competent, as demonstrated by their capacity to secrete IFN-γ in the culture supernatant (Supplementary Fig. 1A) and by their intracellular expression of IFN-γ (Supplementary Fig. 1B).


Deletion of the G6pc2 gene encoding the islet-specific glucose-6-phosphatase catalytic subunit-related protein does not affect the progression or incidence of type 1 diabetes in NOD/ShiLtJ mice.

Oeser JK, Parekh VV, Wang Y, Jegadeesh NK, Sarkar SA, Wong R, Lee CE, Pound LD, Hutton JC, Van Kaer L, O'Brien RM - Diabetes (2011)

Islet autoantigen–specific CD8+ T-cell responses in wild-type and G6pc2-deficient NOD/ShiLtJ mice. A and B: Islets were isolated from individual female wild-type or G6pc2−/− NOD/ShiLtJ mice at 12 weeks of age and cultured in the presence of interleukin-2 for 9 days. Cells were then stained with FITC-labeled anti-CD8α and PE-labeled MHC–class I tetramers loaded with the indicated peptides and analyzed by flow cytometry. Tetramers loaded with Flu-NP366–374 or Flu-NP147–155 were used as a negative control for tetramer staining. Data were electronically gated for CD8α-expressing cells. A: Representative flow cytometry data for one wild-type mouse and one G6pc2-deficient mouse. Numbers indicate the percentage of cells within the gated area. B: Summary of the flow cytometry data for four wild-type and four G6pc2-deficient mice from two experiments. C: Splenic and lymph-node cells were prepared from female wild-type or G6pc2−/− NOD/ShiLtJ mice at 12 weeks of age, and the frequency of NRP-V7–specific CD8+ T cells was determined by tetramer staining. Data were electronically gated for CD8α-expressing cells. Representative flow cytometry data are shown. D: Wild-type or G6pc2−/− NOD/ShiLtJ female mice were immunized with NRP-V7 peptide (100 μg per mouse) emulsified in complete Freund’s adjuvant. Two weeks later, splenic and draining lymph-node cells were prepared and stained with anti–CD8α-FITC and NRP-V7 or control Flu-NP tetramer and analyzed by flow cytometry. Data were electronically gated for CD8α-expressing cells. Representative flow cytometry data are shown. NS, not significant; WT, wild-type. (A high-quality color representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198073&req=5

Figure 4: Islet autoantigen–specific CD8+ T-cell responses in wild-type and G6pc2-deficient NOD/ShiLtJ mice. A and B: Islets were isolated from individual female wild-type or G6pc2−/− NOD/ShiLtJ mice at 12 weeks of age and cultured in the presence of interleukin-2 for 9 days. Cells were then stained with FITC-labeled anti-CD8α and PE-labeled MHC–class I tetramers loaded with the indicated peptides and analyzed by flow cytometry. Tetramers loaded with Flu-NP366–374 or Flu-NP147–155 were used as a negative control for tetramer staining. Data were electronically gated for CD8α-expressing cells. A: Representative flow cytometry data for one wild-type mouse and one G6pc2-deficient mouse. Numbers indicate the percentage of cells within the gated area. B: Summary of the flow cytometry data for four wild-type and four G6pc2-deficient mice from two experiments. C: Splenic and lymph-node cells were prepared from female wild-type or G6pc2−/− NOD/ShiLtJ mice at 12 weeks of age, and the frequency of NRP-V7–specific CD8+ T cells was determined by tetramer staining. Data were electronically gated for CD8α-expressing cells. Representative flow cytometry data are shown. D: Wild-type or G6pc2−/− NOD/ShiLtJ female mice were immunized with NRP-V7 peptide (100 μg per mouse) emulsified in complete Freund’s adjuvant. Two weeks later, splenic and draining lymph-node cells were prepared and stained with anti–CD8α-FITC and NRP-V7 or control Flu-NP tetramer and analyzed by flow cytometry. Data were electronically gated for CD8α-expressing cells. Representative flow cytometry data are shown. NS, not significant; WT, wild-type. (A high-quality color representation of this figure is available in the online issue.)
Mentions: Diabetes in the NOD/ShiLtJ mouse results from the specific destruction of pancreatic β-cells by a cell-mediated process involving an autoreactive antigen-specific CD4+ and CD8+ T-cell attack in an environment where regulatory T-cell responses are perturbed (6). Molecularly defined targets of pathogenic CD8+ T cells have been well established (6) and include an H-2Kd–restricted peptide of G6pc2206–214 and its mimotope NRP-V7 (6) and a H-2Kd–restricted peptide of the insulin B chain (insulin B15–23 and its variant INS-L9), both of which have been implicated in disease pathogenesis and both of which are recognized by an appreciable proportion of CD8+ T cells residing in islet infiltrates in NOD/ShiLtJ mice (6). We compared the prevalence of islet autoantigen-specific CD8+ T cells in islets isolated from 12-week-old wild-type and NOD/ShiLtJ G6pc2−/− mice. Islets were cultured for 9 days ex vivo in the presence of interleukin-2 to expand CD8+ T cells residing in them. Cells were then stained with a panel of peptide-loaded MHC–class I tetramers bearing the abovementioned G6pc2 and insulin peptides, or influenza peptides as negative controls, and analyzed by flow cytometry. As previously reported, CD8+ T cells reactive to NRP-V7 were the dominant population of CD8+ T cells in wild-type mouse islets, accompanied by a variable, lesser percentage of insulin B15–23–and MimA2-specific T cells (Fig. 4A and B). In contrast, G6pc2−/− mouse islets completely lacked CD8+ T cells reactive to NRP-V7 (Fig. 4A and B) but retained a high proportion of CD8+ T cells specific for insulin B (Fig. 4A and B). The CD8+ T cells in the islets of G6pc2−/− mice were functionally competent, as demonstrated by their capacity to secrete IFN-γ in the culture supernatant (Supplementary Fig. 1A) and by their intracellular expression of IFN-γ (Supplementary Fig. 1B).

Bottom Line: Insulitis was prominent in both groups, but whereas NOD/ShiLtJ G6pc2(+/+) islets contained CD8(+) T cells reactive to the G6pc2 NRP peptide, G6pc2 NRP-reactive T cells were absent in NOD/ShiLtJ G6pc2(-/-) islets.These results demonstrate that G6pc2 is an important driver for the selection and expansion of islet-reactive CD8(+) T cells infiltrating NOD/ShiLtJ islets.However, autoreactivity to G6pc2 is not essential for the emergence of autoimmune diabetes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

ABSTRACT

Objective: Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), now known as G6PC2, is a major target of autoreactive T cells implicated in the pathogenesis of type 1 diabetes in both mice and humans. This study aimed to determine whether suppression of G6p2 gene expression might therefore prevent or delay disease progression.

Research design and methods: G6pc2(-/-) mice were generated on the NOD/ShiLtJ genetic background, and glycemia was monitored weekly up to 35 weeks of age to determine the onset and incidence of diabetes. The antigen specificity of CD8(+) T cells infiltrating islets from NOD/ShiLtJ G6pc2(+/+) and G6pc2(-/-) mice at 12 weeks was determined in parallel.

Results: The absence of G6pc2 did not affect the time of onset, incidence, or sex bias of type 1 diabetes in NOD/ShiLtJ mice. Insulitis was prominent in both groups, but whereas NOD/ShiLtJ G6pc2(+/+) islets contained CD8(+) T cells reactive to the G6pc2 NRP peptide, G6pc2 NRP-reactive T cells were absent in NOD/ShiLtJ G6pc2(-/-) islets.

Conclusions: These results demonstrate that G6pc2 is an important driver for the selection and expansion of islet-reactive CD8(+) T cells infiltrating NOD/ShiLtJ islets. However, autoreactivity to G6pc2 is not essential for the emergence of autoimmune diabetes. The results remain consistent with previous studies indicating that insulin may be the primary autoimmune target, at least in NOD/ShiLtJ mice.

Show MeSH
Related in: MedlinePlus