Detection of GAD65-Specific T-Cells by Major Histocompatibility Complex Class II Tetramers in Type 1 Diabetic Patients and At-Risk Subjects

GAD65-induced T-cell activation is heterogenous in at-risk subjects. Six at-risk subjects (A– F) were investigated for the presence of activated CD25⁺-CD4⁺ T-cells on day 3 postrestimulation with immobilized HLA-DR401 or -DR404 monomer containing GAD65 557I peptide. The subjects were 7878 (A), 6212 (B), 6827 (C), 5574 (D), 7657 (E), and 6899 (F). PBMCs from at-risk subjects 7878, 6212, and 6827 (A– C) were obtained at two different time points. Fresh samples from subjects 7878 and 6827 were analyzed at 5 months and those from subject 6212 at 7 months. In all panels cells were gated on a live lymphocyte cell population in forward and side scatter. The cell number in the live population was variable because of the amount of PBMCs available for the analysis. The vertical axis shows CD4 fluorescence, and the horizontal axis shows the CD25 fluorescence. The percentage of CD25⁺/CD4⁺ cells is shown on the upper right quadrant.

Tetramer-binding GAD65-specific cells reside in the CD25⁺/CD4^high+ population. A: An example of the detection of tetramer-positive cells by flow cytometry in a CD25⁺/CD4^high+ cell population in type 1 diabetic patient 7810. B: Staining of cells from at-risk subject 7878, who displayed a distinct CD25⁺/CD4^high+ phenotype upon stimulation by GAD65 peptide. C: Tetramer staining of the GAD65-stimulated cells from another at-risk subject, 7657, who displayed the CD25⁺/CD4^low phenotype. A– C: The left panel represents the staining by a GAD65-557I tetramer and the right panel shows the staining by a control HSV-p61 tetramer. The cells were gated on a live lymphocytic cell population in forward and side scatter and CD25⁺/CD4^high+. The cell number on both gates was variable because of the amount of PBMCs available for the analysis. C: The cells were gated on the top 25% of CD4 staining intensity among the CD25/CD4 double-positive cells because of the lack of a CD25⁺/CD4^high+ cell population in this subject. The vertical axis shows CD4 fluorescence and the horizontal axis shows the tetramer fluorescence. The percentage of tetramer⁺/CD4⁺ cells is shown on the upper right quadrant.

Single-cell-sorted CD25⁺/CD4^high+ cells are GAD65 specific. Cells from an HLA-DR404-positive at-risk subject (6212) were stimulated with GAD65 as described above. Single-cell sorting was performed on the top 1% of the CD4 staining intensity in the cell population gated on forward and side scatter live lymphocytic cell populations and on CD25⁺/CD4^high+ cells. A representative T-cell proliferation of C15, one of seven T-cell clones isolated, is shown. Clone C15 was stimulated by autologous APCs (left chart) and BLS-DR404 B-LCL (right chart) pulsed with 0.1–10 μg/ml GAD65 557I or GAD65 555-567 wild-type (wt) peptide. Each dot represents the mean counts per minute of [³H]thymidine incorporation at 72 h in triplicate cultures. Error bars represent SE