Efficient T cell receptor−mediated apoptosis in nonobese diabetic mouse thymocytes

Supplementary Fig. 1. (pdf 19K)
Immature CD4⁺8⁺ and Semi-mature CD4⁺8^-HSA⁺ thymocytes from NOD mice are sensitive to TCR-CD3-ligation induced apoptosis in vitro. (a) Immature CD4⁺8⁺ and (b) semi-mature CD4⁺8^-HSA⁺ thymocytes from NOD and C57BL/6 mice were purified by immunofluorescent staining with surface marker-specific antibodies and flow cytometric cell sorting. Cells were cultured for 20 h in plates coated with graded concentrations of CD3 mAb in the presence or absence of optimal doses of CD28 mAb. Apoptosis was assessed after 20 h by staining with propidium iodide plus FITC-coupled annexin V followed by flow cytometric analysis. Live cells were PI^-annexin V^-; all other cells were considered dead or dying. Data represent arithmetic means +/- SE of 3-5 independent experiments. Immature CD4⁺8⁺ from NOD mice were more susceptible to spontaneous apoptosis in culture (49 6% within 20 h) than those from C57BL/6 mice (275%; p<0.006).

Supplementary Fig. 2. (pdf 11K)
Immature CD4⁺8⁺ and Semi-mature CD4⁺8^-HSA⁺ thymocytes from NOD mice are sensitive to TCR-CD3-ligation induced apoptosis in vivo. NOD and C57BL/6 mice (6-10 weeks old) were injected i.v. with 20 or 200 g of CD3 mAb (145.2C11) or, as a control, with saline. Animals were sacrificed after 40 h, thymocyte suspensions prepared and total numbers of cells counted. Single cell suspensions of thymocytes were stained with fluorochrome-labeled antibodies to CD4, CD8 and HSA and analyzed by flow cytometry. Total numbers of all thymocytes (not shown), immature CD4⁺8⁺ and semi-mature CD4⁺8^-HSA⁺ thymocytes were calculated by multiplying the total thymic cellularity with the relative percentages of this subset, obtained by immunofluorescent staining with surface marker-specific antibodies and flow cytometric analysis. Data represent arithmetic means + SE of 5 independent experiments with 7-9 animals of each strain and treatment regime. No significant differences were observed in comparing the total numbers of all thymocytes, CD4⁺8⁺ immature thymocytes or CD4⁺8^-HSA⁺ thymocytes between C57BL/6 and NOD mice injected with either 20g CD3 mAb (p=0.243) or 200g CD3 mAb (p=0.596).

Top