DN T cells enhance AICD of proliferated CD4⁺ T cells, and perforin plays a role in DN T-cell–mediated cell death. (A) Annexin V staining of C57BL/6 CD4⁺ T cells stimulated by mature DBA/2 DCs. C57BL/6 DN T cells greatly increased the annexin V⁺ frequency in proliferating C57BL/6 CD4⁺ T cells. In the upper panels, the horizontal bars gate the dividing cells, and the numbers refer to the percentage of these cells; in the lower panels, the horizontal bars gate annexin V staining positive cells, and the numbers refer to the percentages of these cells. (B) CFSE-labeled C57BL/6 (CD45.1) CD4⁺CD25⁻ T cells were stimulated by mature DBA/2 DCs. The suppressor function of C57BL/6 DN T cells converted from wild-type and perforin KKO mice was compared. Results shown here represent 3 independent experiments. The horizontal bars gate the nondividing cells, and the numbers refer to the percentages of these cells. (C) DN T-cell–mediated suppression of CD4⁺CD25⁻ T cell proliferation was attenuated in the absence of perforin. Results shown here are means of 3 independent experiments. (D) Annexin V staining of C57BL/6 CD4⁺CD25⁻ T cells stimulated by mature DBA/2 DCs. DN T cells converted from wild-type, but not perforin KO C57BL/6 mice greatly increased the annexin V⁺ population in proliferating C57BL/6 CD4⁺ T cells.