Increased frequency of CD4⁺FOXP3⁺Helios⁺ T cells in IPEX patients. (A) Representative CD25 and FOXP3 (left) and Helios and FOXP3 staining (right) on CD3⁺CD4⁺ cells from a healthy donor and a representative IPEX patient. CD3⁺CD4⁺CD25⁺CD127^loFOXP3⁺ (B) and CD3⁺CD4⁺FOXP3⁺Helios⁺ (C) T-cell frequencies in 38 healthy donors and 5 IPEX patients. (D) Expression levels of CD25, CD127, and CD45RO on FOXP3⁻Helios⁻ and FOXP3⁺Helios⁺ CD4⁺ T cells from a representative IPEX patient (solid gray) and a healthy donor (bold) gated as shown in panel A. The mean fluorescence intensities (MFI) of (E) CD25, (F) CD127, and (G) CD45RO expression is shown for Helios⁻FOXP3⁻ and Helios⁺FOXP3⁺ CD4⁺ T cells from healthy donors and IPEX patients.