The induction of B-cell apoptosis is specific to activated CD4⁺CD25⁺ T cells. (A) Activated CD4⁺CD25⁺ T cells cause apoptosis of B cells on restimulation. CD4⁺CD25⁺ T cells preactivated for 6 days were cultured with B-cell blasts (preactivated with LPS for 20 hours) at a 5:1 ratio for 8 hours in the presence or absence of anti-CD3 (2 μg/mL). A low amount of LPS (0.5 μg/mL) was also added to the coculture. Annexin V expression and 7-AAD incorporation were evaluated on B220⁺ cells. (B) Activated CD4⁺CD25^– T cells do not induce B-cell death. CD4⁺CD25^– T cells were cultured with B-cell blasts as in panel A, and the cells were stained for B220, annexin V, and 7-AAD. (C) Activated CD4⁺CD25⁺T cells induce B-cell death in response to restimulation with HA peptide. CD4⁺CD25⁺ T cells from HA-TCR transgenic mice were cultured with B-cell blasts as in panel A except that HA peptide (16 μM) was used in place of anti-CD3. (D) Activated CD4⁺CD25⁺ T cells do not induce T-cell apoptosis. CD4⁺CD25^– T cells were stimulated with irradiated T-depleted splenocytes and conA (3 μg/mL) for 20 hours, labeled with CFSE, and cocultured with activated CD4⁺CD25⁺ T cells or with CD8⁺ T cells that had been activated with plate-bound anti-CD3 and IL-2 for 3 days and expanded in IL-2 media for 3 days before use. ConA (3 μg/mL) was added to the cocultures to retarget the CD4⁺CD25⁺ T cells or the activated CD8⁺ T cells to the CD4⁺ blast target cells during the 8-hour coculture. Results are representative of at least 3 experiments.