Carriers of FOXP3 mutations have functionally normal nTregs with exclusive expression of the WT-FOXP3 allele. (A) Expression of FOXP3, CD25, and CD127 in CD4⁺ gated PBMCs derived from the representative carrier 3 (top panel) and a representative healthy female donor (bottom panel). (B) FOXP3 TSDR demethylation levels in genomic DNA samples from whole blood and total PBMCs derived from carriers of FOXP3 mutations and healthy donor females (n = 8). (C) The ability of freshly isolated CD4⁺CD25⁺ T cells (Treg) of carriers 1, 5, and 6 and of a representative healthy donor to suppress activated responder CD4⁺CD45RO⁺ effector T cells (R) is shown. Percentages indicate the inhibition of proliferation. Error bars indicate SD. (D) The pherograms of XCI analyses of sorted CD4⁺CD25^hi (top panel) and CD4⁺CD25⁻ (bottom panel) T cells derived from carriers 1, 3, 5, and 6 (n = 4) and from 1 representative healthy female donor (n = 3) are shown. In CD4⁺CD25^hi T cells derived from carriers of FOXP3 mutations, only the inactive repeats that cosegregate with the mut-FOXP3 allele can be amplified, indicating that only the WT-FOXP3 allele is active. In contrast, in CD4⁺CD25^hi T cells derived from healthy donors (n = 3) both the chromosomes are active. In sorted CD4⁺CD25⁻ T cells derived from carriers of FOXP3 mutations (n = 4) and healthy female donors (n = 3), CAG repeats of both chromosomes can be amplified, indicating that both the WT and the mut alleles are active. For each repeat the cosegregating WT and mut-FOXP3 alleles are indicated. cr indicates carrier; hd, healthy donor.