Figure 2.

Plate-bound Ab stimulation causes apoptotic cell death in CD4⁺CD25⁻ cells. A, CD4⁺ CD25⁻ and CD4⁺CD25⁺ T cells were stimulated with bead-bound or plate-bound anti-CD3 plus anti-CD28 Abs for 5 d and analyzed for Annexin V and 7-AAD staining. The percentage of positive cells in each quadrant is indicated. B, CD4⁺CD25⁻ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 Abs in the presence of IL-2 (10 ng/ml). Cells were harvested at indicated time points and cultured on a new plate devoid of any anti-CD3 or anti-CD28 Abs. On day 5, all of the cells were analyzed for cell death by Annexin V and 7-AAD staining. The numbers represent the percentage of positive cells in the corresponding quadrant. C, Effect of the pan-caspase inhibitor (Z-VAD-fmk) and caspase 3-specific inhibitor (Z-DEVD-fmk) on cell death. CD4⁺CD25⁻ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 Abs. After 2 d, the indicated cultures were supplemented with DMSO, z-VAD-fmk, or no treatment (control). Cell death was determined by Annexin V and 7-AAD staining on day 4. D, DNA ladder analysis of CD4⁺ CD25⁻ T cells stimulated for 5 d with plate-bound or bead-bound anti-CD3 plus anti-CD28 Abs. Total DNA extracted from CD4⁺CD25⁻ T cells stimulated with plate-bound or bead-bound Abs were subjected to electrophoresis. The size of the marker DNA is indicated in the left lane. E, CD4⁺CD25⁻ T cells were stimulated with plate-bound anti-CD3 and anti-CD28 Abs, as in A. After 4 d of stimulation, cells were analyzed for activation of caspase 3 by staining with CaspGLOW staining reagent. Solid lines represent data from stimulated cells, and dotted lines show the data for unstimulated cells. The numbers above the arrow represent the percentage of positive cells from stimulated samples, and the numbers in parentheses show the percentage of positive cells from unstimulated samples.