Expression of FOXP3 and CD95 on T cells derived from cord blood. (A) Freshly isolated adult PBLs as well as cord blood lymphocytes were stained with fluorochrome-labeled mAbs against CD4 and CD25 followed by intracellular staining with anti-FOXP3 mAb (clone PCH101) and were analyzed with a FACS Canto cytometer. Cells were gated on viable CD4⁺ T cells. (B) Adult CD4⁺CD25^hi, cord blood CD4⁺CD25⁺ T cells, and their respective T_conv counterparts were FACS sorted using the indicated gates, and (C) inhibitory capacities of FACS-sorted T_regs were analyzed by proliferation assays. For this purpose, T_convs were FACS sorted from a third-party adult donor and incubated with cord blood T_regs or adult T_regs as described in “Materials and methods.” (D) Freshly isolated adult PBLs as well as cord blood lymphocytes were stained with fluorochrome-labeled mAbs against CD4, CD25, and CD95 followed by intracellular staining with anti-FOXP3 mAb (clone PCH101) and were analyzed on a FACS Canto cytometer. Histogram overlays indicate expression of CD95 on CD4⁺CD25⁺FOXP3⁺ T cells from cord blood versus adult blood. The isotype control for CD95 is shown for cord blood and was similar to isotype staining of adult PBLs. (E) Cord blood lymphocytes were stained as in panel D plus anti-CD45RO mAb. Histogram overlays indicate the expression of CD95 on CD4⁺CD25⁺FOXP3⁺ T cells as well as on CD4⁺CD25⁺FOXP3⁺CD45RO^hi T cells. Isotype control for CD95 staining is shown.