Detection of memory and naive T_regs among human adult CD4⁺CD25⁺FOXP3⁺ T_regs. (A) Freshly isolated adult PBLs were stained directly with fluorochrome-labeled mAbs against CD4, CD25, CD45RO, and CD45RA followed by intracellular staining with anti-FOXP3 mAb (clone PCH101) and were analyzed with a FACS Canto cytometer. After gating for CD4⁺ T cells (R1), CD45RO^hiFOXP3⁺ T cells (R3, blue) as well as CD45RO^loFOXP3⁺ T cells (R2, red) were defined. Identical populations (blue, red) are shown in panels B and C. (B) Multicolor analysis allocates CD45RO^hiFOXP3⁺T cells (blue) as well as CD45RO^loFOXP3⁺ T cells (red) within the CD4⁺CD25⁺ population. (C) CD45RO^hiFOXP3⁺T cells (blue) as well as CD45RO^loFOXP3⁺ T cells (red) were analyzed for expression of CD45RA and compared with T_convs. (D) PBLs were stained with directly fluorochrome-labeled mAbs against CD95, CD4, CD25, CD45RO, and CD45RA followed by intracellular staining with anti-FOXP3 mAb (clone PCH101) to determine expression of CD95 on CD45RO^hiFOXP3⁺ (R3 as in panel B, blue) as well as CD45RO^loFOXP3⁺ (R2 as in panel B, red) cells. The dashed line represents isotype control. (E) Note the significant inverse correlation between the percentage of naive T_regs (CD4⁺CD25⁺FOXP3⁺CD45RO^loCD95^lo T cells) among total T_regs and age. Dots correspond to individual samples tested. The solid line shows linear regression.