Perforin-deficient CD4⁺CD25⁺ T cells are partially deficient in their ability to kill B cells. (A) Induction of perforin expression in CD4⁺CD25^–, CD4⁺CD25⁺, and CD8⁺ T cells after activation. Sorted resting or activated WT CD4⁺CD25^– (⋄), CD4⁺CD25⁺ (♦), CD8⁺ (▴), and perforin-deficient CD4⁺CD25⁺ T cells (○) were collected and analyzed for perforin mRNA expression by quantitative PCR as in Figure 4A. (B) The induction of B-cell death is Ca²⁺ dependent. Activated CD4⁺CD25⁺ T cells were cultured with B-cell blasts from the same mouse at a 5:1 ratio for 8 hours in the presence or absence of anti-CD3. Where indicated, EGTA (3 mM) was included in the culture. The cells were stained for B220, annexin V, and 7-AAD to evaluate B-cell death. Results are expressed as means ± SD (n = 3). (C) Both perforin and granzyme B are required for CD4⁺CD25⁺ T-cell–mediated B-cell apoptosis. Preactivated WT or perforin-deficient CD4⁺CD25⁺ T cells were incubated with B-cell blasts for 8 hours under the conditions specified. Where indicated, CD4⁺CD25⁺ T cells were treated with DCI (30 μM) for 30 minutes and carefully washed before use in the coculture. (D) The suppression of T-cell proliferation is perforin independent. CD4⁺CD25^– responder T cells were stimulated with soluble anti-CD3 and irradiated APCs and cultured with either activated WT (▪) or perforin-deficient (□) CD4⁺CD25⁺ T cells. The proliferation of responder T cells was determined as described in Figure 1A. Results are representative of at least 3 experiments.