Sensitization of cord blood T_regs toward CD95L-mediated cell death in response to TCR stimulation. FACS-sorted cord blood T_regs (CD4⁺CD25⁺) and adult T_regs (CD4⁺CD25^hi) were incubated with anti-CD3/CD28 mAbs and IL-2 as described in “Materials and methods.” CD95 expression (A-B, •) and CD45RO expression (B, ○) were determined at the time points indicated. (C) Six days after TCR stimulation, cord blood T_regs were incubated with CD95L (1:20) for 10 hours, and specific cell death rate was determined and compared with the specific cell death rate of freshly isolated cord blood T_regs. Likewise, freshly isolated FACS-sorted CD45RO^lo cord blood T_regs without CD95^hi memory T_regs (Figure 3B) were incubated with CD95L (1:20) for 10 hours. (D) Proliferation assays were used to measure the inhibitory capacities of freshly isolated cord blood T_regs or 6-day activated T_regs (anti-CD3/CD28 mAbs plus IL-2). For this test, T_convs were FACS sorted from an adult donor and incubated with cord blood T_regs as described in “Materials and methods.” (E-F) Apoptosis-resistant T_regs were incubated with anti-CD3/CD28 mAbs plus IL-2 as described in “Materials and methods.” After 6 days, CD95 sensitivity toward CD95-mediated apoptosis (E) and expression of CD95 (F) were determined. One representative experiment of at least 3 independent experiments is shown. Error bars represent SEM.