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Immunology

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

  1. Helena Reijonen,
  2. Erik J. Novak,
  3. Sharon Kochik,
  4. Anne Heninger,
  5. Andrew W. Liu,
  6. William W. Kwok and
  7. Gerald T. Nepom
  1. From the Virginia Mason Research Center, Benaroya Research Institute, Seattle, Washington
    Diabetes 2002 May; 51(5): 1375-1382. https://doi.org/10.2337/diabetes.51.5.1375
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    • FIG. 1.
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      FIG. 1.

      GAD65-stimulated cells display CD25+/CD4high+ phenotype in type 1 diabetic patients. A, C, and D: Flow cytometry analysis of cells (obtained from newly diagnosed type 1 diabetic patients) that were restimulated with GAD65 557I peptide on HLA-DR401 monomer. A: Patient 7810. C: Patient 7826 D: Patient 7858. B: Flow cytometry analysis of cells obtained from a newly diagnosed type 1 diabetic patient (7929) that were restimulated with GAD65 557I peptide on DR404 monomer. In all panels, cells were gated on live lymphocyte cell populations 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. HLA-DR401+ normal subjects are shown in E (1010) and F (7842).

    • FIG. 2.
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      FIG. 2.

      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.

    • FIG. 3.
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      FIG. 3.

      Tetramer-binding GAD65-specific cells reside in the CD25+/CD4high+ population. A: An example of the detection of tetramer-positive cells by flow cytometry in a CD25+/CD4high+ cell population in type 1 diabetic patient 7810. B: Staining of cells from at-risk subject 7878, who displayed a distinct CD25+/CD4high+ phenotype upon stimulation by GAD65 peptide. C: Tetramer staining of the GAD65-stimulated cells from another at-risk subject, 7657, who displayed the CD25+/CD4low 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+/CD4high+. 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+/CD4high+ 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.

    • FIG. 4.
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      FIG. 4.

      Single-cell-sorted CD25+/CD4high+ 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+/CD4high+ 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

    • FIG. 5.
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      FIG. 5.

      Single-cell-sorted CD25+/CD4high+ cells are GAD65 tetramer-positive. Cells from an HLA-DR401-positive at-risk subject (5574) were stimulated with GAD65 as described above. Single-cell sorting was performed on the top 1% of CD4 staining intensity in the CD25+ live lymphocytic cell population. Clone 164 was stained by GAD65 557I tetramer (left panel) or HSV-p61 control tetramer (right panel). The vertical axis shows CD4 fluorescence, and the horizontal axis shows the tetramer fluorescence.

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    • TABLE 1

      CD25+/CD4high+ phenotype and detection of tetramer-positive cells in type 1 diabetic patients and at-risk and normal subject

      SubjectCD4high+/CD25+CD4+/CD25+*Tetramer staining†Type 1 diabetes
      7810Yes10.928.7Yes
      7929Yes11.525.0Yes
      7826Yes10.74.1Yes
      7858Yes4.35.0Yes
      6212Yes7.48.6At risk
      7878Yes8.75.2At risk
      5574Yes18.05.4At risk
      7657No24.7<1At risk
      6827No2.1<1At risk
      6899No0.2<1At risk
      7877No2.0<1No
      7842No0.3<1No
      1010No4.7<1No
      7029No0.4<1No
      3116No1.7<1No
      • Data are %. Number of CD25+/CD4+ cells in total cell population gated on the live lymphocyte cell population in forward and side scatter;

      • †

        † number of tetramer-positive cells in total cell population gated on the live lymphocyte cell population in forward and side scatter and on CD25+/CD4high+.

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    Detection of GAD65-Specific T-Cells by Major Histocompatibility Complex Class II Tetramers in Type 1 Diabetic Patients and At-Risk Subjects
    Helena Reijonen, Erik J. Novak, Sharon Kochik, Anne Heninger, Andrew W. Liu, William W. Kwok, Gerald T. Nepom
    Diabetes May 2002, 51 (5) 1375-1382; DOI: 10.2337/diabetes.51.5.1375

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    Detection of GAD65-Specific T-Cells by Major Histocompatibility Complex Class II Tetramers in Type 1 Diabetic Patients and At-Risk Subjects
    Helena Reijonen, Erik J. Novak, Sharon Kochik, Anne Heninger, Andrew W. Liu, William W. Kwok, Gerald T. Nepom
    Diabetes May 2002, 51 (5) 1375-1382; DOI: 10.2337/diabetes.51.5.1375
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