T_reg reconstitution in a representative patient (UPN 301). T_regs were determined by flow and qRT-PCR analysis before and 30, 60, and 90 days after transplantation (Pre, d30, d60, d90, respectively); donor (Don) and the CD25-depleted lymphocyte product (SD). (A) Flow analysis for FOXP3 and CD25 in the CD4⁺ subset. Percentages displayed represent the fraction of positive cells in CD4⁺ cells. Two T_reg populations, a CD25⁺ CD4⁺ FOXP3⁺ and a CD25⁻ CD4⁺ FOXP3⁺ subset, could be identified before and after transplantation as well in the donor. The SD product was entirely CD25⁻ but contained a CD25⁻ CD4⁺ FOXP3⁺ subset of T_regs. CD25⁻ CD4⁺ FOXP3⁺ and CD25⁺ CD4⁺ FOXP3⁺ T_reg fractions increased after SCT. CD25⁺ CD4⁺ FOXP3⁻ T_effs declined after SCT. (B) Total fraction of FOXP3⁺ cells in CD4⁺ cells. (C) Foxp3 mRNA expression in CD4-selected cells expressed as copy numbers per 1 × 10⁶ copies of β-actin.