Figure 6.

Apoptosis caused by plate-bound Abs is dependent on p53. A, CD4⁺CD25⁻ (upper panels) and CD4⁺CD25⁻ (lower panels) T cells from WT (left panels) and p53^−/− C57.BL/6 (right panels) mice were stimulated with plate-bound anti-CD3 and anti-CD28 Abs for 5 d and analyzed for cell death by Annexin V and 7-AAD staining. B, Total number of live cells recovered on indicated days after stimulation of CD4⁺CD25⁻ (circles) and CD4⁺ CD25⁺ (squares) T cells from WT B6 (open symbols) and p53^−/− (filled symbols) mice. C, Allogenic DCs cause p53-dependent T cell death. Sorted CD4⁺CD25⁻ and CD4⁺ CD25⁺ T cells from B6 and p53^−/− (B6 background) mice were stimulated with sorted CD11c⁺ cells from BALB/c mice (2.5 × 10³ T cells/well and 4 × 10³ DCs per well in U-bot-tom 96-well plates in the presence of IL-2). At the indicated time points, cells were harvested, and viable cells were analyzed by analysis of trypan blue exclusion and FoxP3 expression. D, The live cell numbers of FoxP3⁻ and FoxP3⁺ T cells in CD4⁺CD25⁺ cultures. The number of FoxP3⁺ cells was calculated based on total live cells and the percentage of FoxP3⁺ T cells present in the culture. At the start of the culture, WT and p53^−/− CD4⁺CD25⁺ T cell populations contained 92% and 91% FoxP3⁺ cells, respectively. E, Fas expression by CD4⁺CD25⁻ cells and CD4⁺CD25⁺ cells from WT C57.BL/6 (upper panels) and p53^−/− mice (lower panels). Fas expression by un-stimulated cells (dotted lines) and cells stimulated with plate-bound anti-CD3 plus anti-CD28 Abs (solid lines) are shown. Numbers represent the percentage of Fas-positive cells for stimulated and unstimulated (parenthesized) cells.