Diabetes-Specific HLA-DR–Restricted Proinflammatory T-Cell Response to Wheat Polypeptides in Tissue Transglutaminase Antibody–Negative Patients With Type 1 Diabetes
Article Figures & Tables
Figures
- FIG. 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.
- FIG. 2.
T-cell proliferation in response to WPs and other antigens or mitogen. 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 WP, ovalbumin (OVA), gliadin, 33-mer, insulin, tetanus toxoid (TT), or phytohemagglutinin (PHA) (see research design and methods for details). On day 8, cells were stained with Cy-chrome–conjugated anti-CD3 monoclonal antibody. CDI was calculated. A CDI value greater than the control mean + 3 SD (CDI >14.6) was used to define a positive response to WP. P values indicate statistical difference compared with control subjects. The horizontal lines indicate the means.
- FIG. 3.
WP-stimulated cytokine secretion by PBMNC at day 8 of culture. Box and Whisker plots with individual values for control and diabetic subjects. IFN-γ, TNF, IL-6, IL-4, and IL-10 were measured by flow CBA. IL-17A was measured by ELISA. Each plot includes the mean (dashed line), median (solid line), distribution, and range. (For IL-4, the difference between control and type 1 diabetic [T1D] subjects is significant when the circled outlier value in the control group is removed [P = 0.03].)
- FIG. 4.
Graphic representation of the relationship between wheat-induced T-cell response and high-resolution HLA diabetes risk alleles. Box and Whisker plots with individual values for diabetic subjects. Each plot includes the mean (bold line), median (solid thin line), distribution, and range. A: Distribution of T-cell proliferation (CDI) to WP in type 1 diabetic patients with HLA-DRB1*03 and HLA-DRB1*04 or in those heterozygous for HLA-DRB1*03/*04. A CDI value greater than mean + 3 SD of the control group (CDI >14.6, dashed line) was used to define a positive proliferation response. One of the type 1 diabetic patients with HLA-DRB1*03+/*04− showed positive responses to WP (○). Type 1 diabetic patients who carried one risk allele HLA-DRB1*04 (10 of 20) or patients who carried both risk genes HLA-DRB1*03/*04 (9 of 12) showed positive reactivity to WP. High-resolution results of HLA-DR are labeled beside each individual. B: Distribution of T-cell proliferation (CDI) to WP in type 1 diabetic patients with HLA-DQB1*02 and HLA-DQB1*03 or in those heterozygous for HLA-DQB1*02/*03. High-resolution analyses for HLA-DQB1*02 and *03 are labeled beside each symbol. ↓ (beside 0201) means all individuals in the column carry HLA-DQB1*0201. (Three of the 42 type 1 diabetic individuals are neither DR3 nor DR4, and therefore they are not included in this figure. One of the seven individuals with DR3/non-DR4 in the left panel is heterozygous for DQ*02/*03 and was therefore placed in the middle column in the right panel [DQ*02*03]. Differences in high-resolution haplotypes between responders and nonresponders were not evaluated statistically because of the small sample size.)
- FIG. 5.
Role of HLA-DR in the type 1 diabetes T-cell and cytokine response to WPs. A: The T-cell response to wheat protein (6.2 μg/ml) was calculated as CDI in 14 patients and compared with cells cultured in the presence of anti–HLA-DR monoclonal antibody (5 μg/ml) or with isotype control antibody. B: Cytokine profiles of supernatants from WP-stimulated PBMNC of patients were evaluated in the absence and presence of monoclonal HLA-DR antibodies using Th1/Th2 CBAs for IFN-γ, IL-4, TNF, IL-10, and IL-6 or an ELISA kit for IL-17A.
Tables
- TABLE 1
Description of control subjects and patients with type 1 diabetes
Group n Sex ratio (female/male) Age Duration of diabetes (years) Type 1 diabetic patients 42 28/14 26.7 (8–41) 11.2 ± 7 Healthy control subjects 22 11/11 24.5 (18–32) NA -
Data are means (range) or means ± SD unless otherwise indicated. NA, not applicable.
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- TABLE 2
HLA-DR and -DQ in type 1 diabetic responders and nonresponders
n DR3 DR4 DR3/DR4 DQ2 DQ3 Nonresponders 22 41 59 14 41 64 Responders 20 50 95 (P = 0.01) 45 (P = 0.04) 50 (P = 0.75) 95 (P = 0.02) -
Data are % unless otherwise indicated. T-cell proliferation (CDI) to WP was evaluated in type 1 diabetic patients. Patients with a CDI value greater than mean + 3 SD of the control group (CDI >14.6) were classified as responders. Those below were classified as nonresponders. The percentage of individuals with the indicated HLA haplotypes in these groups was calculated.
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