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Diabetes-specific HLA-DR-restricted proinflammatory T-cell response to wheat polypeptides in tissue transglutaminase antibody-negative patients with type 1 diabetes.

Mojibian M, Chakir H, Lefebvre DE, Crookshank JA, Sonier B, Keely E, Scott FW - Diabetes (2009)

Bottom Line: HLA-DQ2, the major celiac disease risk gene, was not significantly different.T-cell reactivity to WPs was frequently present in type 1 diabetic patients and associated with HLA-DR4 but not HLA-DQ2.The presence of an HLA-DR-restricted Th1 and Th17 response to WPs in a subset of patients indicates a diabetes-related inflammatory state in the gut immune tissues associated with defective oral tolerance and possibly gut barrier dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Canada.

ABSTRACT

Objective: There is evidence of gut barrier and immune system dysfunction in some patients with type 1 diabetes, possibly linked with exposure to dietary wheat polypeptides (WP). However, questions arise regarding the frequency of abnormal immune responses to wheat and their nature, and it remains unclear whether such responses are diabetes specific.

Research design and methods: In type 1 diabetic patients and healthy control subjects, the immune response of peripheral CD3(+) T-cells to WPs, ovalbumin, gliadin, alpha-gliadin 33-mer peptide, tetanus toxoid, and phytohemagglutinin was measured using a carboxyfluorescein diacetate succinimidyl ester (CFSE) proliferation assay. T-helper cell type 1 (Th1), Th2, and Th17 cytokines were analyzed in WP-stimulated peripheral blood mononuclear cell (PBMNC) supernatants, and HLA was analyzed by PCR.

Results: Of 42 patients, 20 displayed increased CD3(+) T-cell proliferation to WPs and were classified as responders; proliferative responses to other dietary antigens were less pronounced. WP-stimulated PBMNCs from patients showed a mixed proinflammatory cytokine response with large amounts of IFN-gamma, IL-17A, and increased TNF. HLA-DQ2, the major celiac disease risk gene, was not significantly different. Nearly all responders carried the diabetes risk gene HLA-DR4. Anti-DR antibodies blocked the WP response and inhibited secretion of Th1 and Th17 cytokines. High amounts of WP-stimulated IL-6 were not blocked.

Conclusions: T-cell reactivity to WPs was frequently present in type 1 diabetic patients and associated with HLA-DR4 but not HLA-DQ2. The presence of an HLA-DR-restricted Th1 and Th17 response to WPs in a subset of patients indicates a diabetes-related inflammatory state in the gut immune tissues associated with defective oral tolerance and possibly gut barrier dysfunction.

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Antigen-specific CD3+ T-cell proliferation. 1.2 × 106 CFSE-labeled PBMNCs from patients with type 1 diabetes (T1D) or healthy control subjects were cultured for 8 days in the absence or presence of different concentrations of WPs. On day 8, cells were stained with Cy-chrome conjugated anti-CD3 monoclonal antibody. CDI was calculated based on a fixed number of 5,000 CD3+ CFSEbright cells using the following formula: CDI = number of CD3+, CFSEdim cells with antigen/number of CD3+, CFSEdim cells without antigen (medium). The horizontal line indicates the mean.
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Figure 1: Antigen-specific CD3+ T-cell proliferation. 1.2 × 106 CFSE-labeled PBMNCs from patients with type 1 diabetes (T1D) or healthy control subjects were cultured for 8 days in the absence or presence of different concentrations of WPs. On day 8, cells were stained with Cy-chrome conjugated anti-CD3 monoclonal antibody. CDI was calculated based on a fixed number of 5,000 CD3+ CFSEbright cells using the following formula: CDI = number of CD3+, CFSEdim cells with antigen/number of CD3+, CFSEdim cells without antigen (medium). The horizontal line indicates the mean.

Mentions: The CFSE assay permitted evaluation of T-cell proliferation to low, nontoxic concentrations of WP (Fig. 1). CD3+ T-cell proliferation in the presence of 3.1 and 6.2 μg/ml WP was significantly higher in the type 1 diabetic patient group compared with control subjects (Fig. 1). At 12.4 μg/ml, CDI was still higher in the type 1 diabetic patient group but the response was less than at 6.2 μg/ml, suggesting inhibition. Therefore, 6.2 μg/ml was chosen as the optimum concentration. To identify patients with a positive proliferation response to WP, the mean + 3 SD of the control group CDI (CDI ≥14.6) was chosen as the cutoff. Of the 42 patients with type 1 diabetes, 20 (47%) had a positive proliferation response to WP. In an expanded analysis of two responders, the proliferative response to WP was mainly from CD4+ T-cells with only a weak CD8+ T-cell response (data not shown). The distribution of CDI to WP was significantly different between patients (n = 42) and control subjects (n = 22); for patients, the median was 11.8 (range 1.0–323) and for control subjects 4.5 (0.8–12.8) (P = 0.0004; Mann-Whitney U test) (Fig. 1).


Diabetes-specific HLA-DR-restricted proinflammatory T-cell response to wheat polypeptides in tissue transglutaminase antibody-negative patients with type 1 diabetes.

Mojibian M, Chakir H, Lefebvre DE, Crookshank JA, Sonier B, Keely E, Scott FW - Diabetes (2009)

Antigen-specific CD3+ T-cell proliferation. 1.2 × 106 CFSE-labeled PBMNCs from patients with type 1 diabetes (T1D) or healthy control subjects were cultured for 8 days in the absence or presence of different concentrations of WPs. On day 8, cells were stained with Cy-chrome conjugated anti-CD3 monoclonal antibody. CDI was calculated based on a fixed number of 5,000 CD3+ CFSEbright cells using the following formula: CDI = number of CD3+, CFSEdim cells with antigen/number of CD3+, CFSEdim cells without antigen (medium). The horizontal line indicates the mean.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2712773&req=5

Figure 1: Antigen-specific CD3+ T-cell proliferation. 1.2 × 106 CFSE-labeled PBMNCs from patients with type 1 diabetes (T1D) or healthy control subjects were cultured for 8 days in the absence or presence of different concentrations of WPs. On day 8, cells were stained with Cy-chrome conjugated anti-CD3 monoclonal antibody. CDI was calculated based on a fixed number of 5,000 CD3+ CFSEbright cells using the following formula: CDI = number of CD3+, CFSEdim cells with antigen/number of CD3+, CFSEdim cells without antigen (medium). The horizontal line indicates the mean.
Mentions: The CFSE assay permitted evaluation of T-cell proliferation to low, nontoxic concentrations of WP (Fig. 1). CD3+ T-cell proliferation in the presence of 3.1 and 6.2 μg/ml WP was significantly higher in the type 1 diabetic patient group compared with control subjects (Fig. 1). At 12.4 μg/ml, CDI was still higher in the type 1 diabetic patient group but the response was less than at 6.2 μg/ml, suggesting inhibition. Therefore, 6.2 μg/ml was chosen as the optimum concentration. To identify patients with a positive proliferation response to WP, the mean + 3 SD of the control group CDI (CDI ≥14.6) was chosen as the cutoff. Of the 42 patients with type 1 diabetes, 20 (47%) had a positive proliferation response to WP. In an expanded analysis of two responders, the proliferative response to WP was mainly from CD4+ T-cells with only a weak CD8+ T-cell response (data not shown). The distribution of CDI to WP was significantly different between patients (n = 42) and control subjects (n = 22); for patients, the median was 11.8 (range 1.0–323) and for control subjects 4.5 (0.8–12.8) (P = 0.0004; Mann-Whitney U test) (Fig. 1).

Bottom Line: HLA-DQ2, the major celiac disease risk gene, was not significantly different.T-cell reactivity to WPs was frequently present in type 1 diabetic patients and associated with HLA-DR4 but not HLA-DQ2.The presence of an HLA-DR-restricted Th1 and Th17 response to WPs in a subset of patients indicates a diabetes-related inflammatory state in the gut immune tissues associated with defective oral tolerance and possibly gut barrier dysfunction.

View Article: PubMed Central - PubMed

Affiliation: Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Canada.

ABSTRACT

Objective: There is evidence of gut barrier and immune system dysfunction in some patients with type 1 diabetes, possibly linked with exposure to dietary wheat polypeptides (WP). However, questions arise regarding the frequency of abnormal immune responses to wheat and their nature, and it remains unclear whether such responses are diabetes specific.

Research design and methods: In type 1 diabetic patients and healthy control subjects, the immune response of peripheral CD3(+) T-cells to WPs, ovalbumin, gliadin, alpha-gliadin 33-mer peptide, tetanus toxoid, and phytohemagglutinin was measured using a carboxyfluorescein diacetate succinimidyl ester (CFSE) proliferation assay. T-helper cell type 1 (Th1), Th2, and Th17 cytokines were analyzed in WP-stimulated peripheral blood mononuclear cell (PBMNC) supernatants, and HLA was analyzed by PCR.

Results: Of 42 patients, 20 displayed increased CD3(+) T-cell proliferation to WPs and were classified as responders; proliferative responses to other dietary antigens were less pronounced. WP-stimulated PBMNCs from patients showed a mixed proinflammatory cytokine response with large amounts of IFN-gamma, IL-17A, and increased TNF. HLA-DQ2, the major celiac disease risk gene, was not significantly different. Nearly all responders carried the diabetes risk gene HLA-DR4. Anti-DR antibodies blocked the WP response and inhibited secretion of Th1 and Th17 cytokines. High amounts of WP-stimulated IL-6 were not blocked.

Conclusions: T-cell reactivity to WPs was frequently present in type 1 diabetic patients and associated with HLA-DR4 but not HLA-DQ2. The presence of an HLA-DR-restricted Th1 and Th17 response to WPs in a subset of patients indicates a diabetes-related inflammatory state in the gut immune tissues associated with defective oral tolerance and possibly gut barrier dysfunction.

Show MeSH
Related in: MedlinePlus