Resistance to CD95L-mediated apoptosis of a T_reg subpopulation. (A) Isolation of apoptosis-resistant T_regs (rT_regs). FACS-sorted T_regs were incubated with CD95L (1:20), and rT_regs were recovered after removal of dead cells using annexin V microbeads as described in “Materials and methods.” (B) rT_regs were incubated with different concentrations of CD95L, and specific cell death rates were compared with those of T_regs cultured in control medium. (C-D) FACS-sorted T_regs (CD4⁺CD25^hi) were incubated alone (C) or with CD95L (1:20) (D), and the remaining cells were analyzed for the expression of CD25 and CD45RO. (E) The percentage of CD45RO^lo and CD45RO^hi subpopulations among the total surviving cell population was determined for various concentrations of CD95L.