Table 1.
List of DQ8-restricted GAD65 peptides used in this study.
Figure 1.
Detection of DQ8-specific self-reactive T cells in T1D patients.
CD4+ T cells from T1D patients were stimulated with self-peptides derived from GAD65. The cells were cultured for two weeks and stained with PE-tetramers. CD4+ T cell responses to GAD65121–140 and GAD65250–266 were detected in multiple subjects. Representative tetramer staining of DQ8-specific self-reactive T cells was shown. Left column: negative staining from a healthy control. Middle and right columns: positive staining from two different T1D patients. Subjects were considered tetramer-positive if a distinct population that was more than three-fold above background in the same experiment was labeled.
Table 2.
Prevalence of GAD65-specific T cells in T1D and healthy subjects.
Figure 2.
Functional analysis of DQ8-specific self-reactive T cells.
T cell clones isolated by DQ8 tetramers were assayed for specificity and functionality. (A) Tetramer staining for GAD65121–140-specific (clone T1D05-C2), GAD65250–266-specific (clone T1D04-C1) and unrelated T cell clones. (B) Representative proliferation results of one GAD65121–140- and two GAD65250–266-specific T cell clones using APCs from a DR0401/DQ8 homozygous individual. Cells were stimulated with specific or irrelevant control peptide in the absence or presence of 20 µg/ml of L243 (HLA-DR blocking antibody) or SPVL3 (HLA-DQ blocking antibody). SI: stimulation index; cpm of specific peptide divided by cpm of irrelevant peptide. (C) Summary of cytokine-positive clones (for definition see Materials and methods) for GAD65121–140 and GAD65250–266. T cell clones were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate and 1 µg/mL ionomycin in the presence of 10 µg/mL Brefeldin A in 1 mL of T cell medium for 4 hours at 37°C. Cells were fixed, permeabilized, stained with antibodies for IFN-γ, IL-10, IL-4, and IL-17, and analyzed on a LSRII multicolor flow cytometer. (D) Secretion of IFN-γ, IL-4, and IL-10 from two GAD65121-140-specific and two GAD65250–266-specific T cell clones. Clones were stimulated in the presence of 10 µg/ml of specific peptide with DQ8 antigen presenting cells.
Figure 3.
Direct ex vivo analysis of GAD65-specific T cells.
Unmanipulated PBMCs were stained with DQ8/GAD65250–266 PE-tetramer. Antigen-specific CD4+ T cells were enriched, stained with antibodies against surface markers of interest, and analyzed on a Calibur multi-color flow cytometer. (A) Representative ex vivo analysis of the surface memory marker CD45RO for GAD65250–266. The frequency of GAD65250–266-specific CD45RO+CD4+ T cells was 4.4 per million CD4+ T cells for the T1D patient (left panel) and 0.6 per million for the healthy subject (right panel). (B) Ex vivo co-staining of GAD65250–266-specific cells with DQ8/GAD65250–266 PE- and DQ8/GAD65250–266 APC-tetramers. Cells were stained with PE-labeled DQ8/GAD65250–266 tetramers first. After enrichment, tetramer-positive cells were stained again with APC-labeled DQ8/GAD65250–266 tetramers at 37°C for 1 h. (C) Cumulative total CD4+ and CD45RO+CD4+ T cell frequencies for GAD65250–266 in controls (open circles, n = 10) and T1D patients (closed circles, n = 10). *** P<0.001, ** P<0.01, as evaluated by Mann-Whitney U-test.
Table 3.
Summary of ex vivo results for GAD65250–266.